Effect of the Structure of Oxypropylated Hydroxyl-Containing Molecules on Glycolysis of Foamed Polyurethane

The activity of a polyether derived from ethylene and propylene oxides (Laprol 5003) and of N,N,N',N'-tetra(hydroxypropyl)ethylenediamine (Lapramol 294) in glycolytic degradation of elastic foamed polyurethane was studied.     

6-Arylamino-3-methyl-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines: Novel N-Arylamidine Structures

We propose a method for obtaining derivatives of 7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine by alkylation of 4-amino-5-methyl-4H-1,2,4-triazole-3-thiol by substituted -chloroacetanilides, followed by cyclization of the intermediate by phosphorus oxychloride.     

2a,4a-Diazacyclopenta[c,d]Azulenes

A method of preparing 5-aryl-1,2-pentaniethyleneimidazoles from O-methylcaprolactim and -aminoacetophenone hydrochlorides is described. It is shown that 1, 2-tri-, -tetra-, and -pentamethyleneirrüdazoles and phenacyl bromide give the corresponding quaternary imidazolium salts, cyclization of which in the presence of base gives the previously unknown 5,6, 78-tetrahydro-2a, 4a-diazacyclopenta(c,dJazulenes (IVa-i).T. G. Shevchenko National Unkiversity, Kiev 254017. T. G. Shevchenko Educational Institute, Chernigov 250037. Translated from Khimiya Geterotskilicheskikh Soedinenii, No. 8, pp. 1072–1077, August, 1996. Original article submitted February 27, 1996; revision submitted June 27, 1996.     

Automated linkage of proteins and payloads producing monodisperse conjugates

Controlled protein functionalization holds great promise for a wide variety of applications. However, despite intensive research, the stoichiometry of the functionalization reaction remains difficult to control due to the inherent stochasticity of the conjugation process. Classical approaches that exploit peculiar structural features of specific protein substrates, or introduce reactive handles via mutagenesis, are by essence limited in scope or require substantial protein reengineering. We herein present equimolar native chemical tagging (ENACT), which precisely controls the stoichiometry of inherently random conjugation reactions by combining iterative low-conversion chemical modification, process automation, and bioorthogonal trans-tagging. We discuss the broad applicability of this conjugation process to a variety of protein substrates and payloads.

Controlled protein functionalization holds great promise for a wide variety of applications.

Applications of protein conjugates are limitless, including imaging, diagnostics, drug delivery, and sensing.^1–4 In many of these applications, it is crucial that the conjugates are homogeneous.⁵ The site-selectivity of the conjugation process and the number of functional labels per biomolecule, known as the degree of conjugation (DoC), are crucial parameters that define the composition of the obtained products and are often the limiting factors to achieving adequate performance of the conjugates. For instance, immuno-PCR, an extremely sensitive detection technique, requires rigorous control of the average number of oligonucleotide labels per biomolecule (its DoC) in order to achieve high sensitivity.⁶ In optical imaging, the performance of many super-resolution microscopy techniques is directly defined by the DoC of fluorescent tags.⁷ For therapeutics, an even more striking example is provided by antibody–drug conjugates, which are prescribed for the treatment of an increasing range of cancer indications.⁸ A growing body of evidence from clinical trials indicates that bioconjugation parameters, DoC and DoC distribution, directly influence the therapeutic index of these targeted agents and hence must be tightly controlled.⁹Standard bioconjugation techniques, which rely on nucleophile–electrophile reactions, result in a broad distribution of different DoC species (Fig. 1a), which have different biophysical parameters, and consequently different functional properties.¹⁰Open in a separate windowFig. 1Schematic representation of the types of protein conjugates.To address this key issue and achieve better DoC selectivity, a number of site-specific conjugation approaches have been developed (Fig. 1b). These techniques rely on protein engineering for the introduction of specific motifs (e.g., free cysteines,¹¹ selenocysteines,¹² non-natural amino acids,^13,14 peptide tags recognized by specific enzymes^15,16) with distinct reactivity compared to the reactivity of the amino acids present in the native protein. These motifs are used to simultaneously control the DoC (via chemo-selective reactions) and the site of payload attachment. Both parameters are known to influence the biological and biophysical parameters of the conjugates,¹¹ but so far there has been no way of evaluating their impact separately.The influence of DoC is more straightforward, with a lower DoC allowing the minimization of the influence of payload conjugation on the properties of the protein substrate. The lowest DoC that can be achieved for an individual conjugate is 1 (corresponding to one payload attached per biomolecule). It is noteworthy that DoC 1 is often difficult to achieve through site-specific conjugation techniques due to the symmetry of many protein substrates (e.g., antibodies). Site selection is a more intricate process, which usually relies on a systematic screening of conjugation sites for some specific criteria, such as stability or reactivity.¹⁷Herein, we introduce a method of accessing an entirely new class of protein conjugates with multiple conjugation sites but strictly homogenous DoCs (Fig. 1c). To achieve this, we combined (a) iterative low conversion chemical modification, (b) process automation, and (c) bioorthogonal trans-tagging in one workflow.The method has been exemplified for protein substrates, but it is applicable to virtually any native bio-macromolecule and payload. Importantly, this method allows for the first time the disentangling of the effects of homogeneous DoC and site-specificity on conjugate properties, which is especially intriguing in the light of recent publications revealing the complexity of the interplay between payload conjugation sites and DoC for in vivo efficacy of therapeutic bioconjugates.¹⁸ Finally, it is noteworthy that this method can be readily combined with an emerging class of site-selective bioconjugation reagents to produce site-specific DoC 1 conjugates, thus further expanding their potential for biotechnology applications.¹⁹     

The cell-penetrating peptide TAT(48-60) induces a non-lamellar phase in DMPC membranes.

Cell-penetrating peptides (CPPs) are short polycationic sequences that can translocate into cells without disintegrating the plasma membrane. CPPs are useful tools for delivering cargo, but their molecular mechanism of crossing the lipid bilayer remains unclear. Here we study the interaction of the HIV-derived CPP TAT (48-60) with model membranes by solid-state NMR spectroscopy and electron microscopy. The peptide induces a pronounced isotropic (31)P NMR signal in zwitterionic DMPC, but not in anionic DMPG bilayers. Octaarginine and to a lesser extent octalysine have the same effect, in contrast to other cationic amphiphilic membrane-active peptides. The observed non-lamellar lipid morphology is attributed to specific interactions of polycationic peptides with phosphocholine head groups, rather than to electrostatic interactions. Freeze-fracture electron microscopy indicates that TAT(48-60) induces the formation of rodlike, presumably inverted micelles in DMPC, which may represent intermediates during the translocation across eukaryotic membranes.     

New 3-hydroxyflavone derivatives for probing hydrophobic sites in microheterogeneous systems

Nine new 3-hydroxyflavone derivatives as fluorescent molecular sensors having two well-separated emission bands were synthesized. These sensors can occupy well-determined locations and orientations in macromolecular ensembles, such as micelles due to their finely-tuned designs. These polarity-sensitive dyes can incorporate into the anhydrous hydrophobic core of aqueous micelles.     

Use of Tetramethylthiuram Disulfide in Synthesis of Nitrogen-containing Heterocyclic Compounds

We have developed a method for synthesis of aryl isothiocyanates by means of thiocarbamoylation of aromatic amines by tetramethylthiuram disulfide followed by degradation of the intermediate N₍₁₎-aryl-N,N-dimethylthiourea by concentrated HCl. We have shown that thiocarbamoylation of 4-amino-5-ethyl-4H-1,2,4-triazole-3-thiol occurs at the 2 position of the triazole ring, while thiocarbamoylation of 4-amino-3-methyl-6-phenyl-4,5-dihydro-1,2,4-triazin-5-one leads to the dihetaryl-substituted thiourea. We consider the possibility of using N₍₁₎-aryl-N,N-dimethylthioureas as analogs of isothiocyanates in reactions with N-nucleophiles.__________Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 5, pp. 775–780, May, 2005.     

Synthesis and Transformations of 3-Ethoxycarbonyl-2-(N-R-thioureido)thiophenes

3-Ethoxycarbonyl-2-(N-R-thioureido)-4,5,6,7-tetrahydrobenzo[b]thiophenes were obtained by the reaction of 2-amino-3-ethoxycarbonyl-4,5,6,7-tetrahydrobenzo[b]thiophene with isothiocyanates and of 3-ethoxycarbonyl-2-isothiocyanato-4,5,6,7-tetrahydrobenzo[b]thiophene with primary and secondary amines. The cyclization paths of the products leading to derivatives of thieno[2,3-d]pyrimidine and thieno[2,3-d]-1,3-thiazine were studied. The corresponding S-substituted derivatives were obtained by the alkylation of 3-R-2-thioxo-3,4,5,6,7,8-hexahydrobenzo[b]thieno[2,3-d]pyrimidin-4-ones.     

Ultrasensitive fluorescent probe for the hydrophobic range of solvent polarities

The new 3-hydroxychromone derivative 2-(6-diethylaminobenzo[b]furan-2-yl)-3-hydroxychromone (FA) displays a dramatic solvent-dependent transformation of fluorescence spectra in the range of low-polarity solvents. The two well-separated emission bands change their relative intensities so that the short-wavelength band being of a very low intensity in hexane becomes dominant in the more polar ethyl acetate and trichloromethane. We suggest the participation in this effect of excited-state intramolecular proton transfer, which is characteristic for other 3-hydroxychromone and 3-hydroxyflavone derivatives, in the range of solvents of much higher polarities. Because of these unique properties, a number of spectroscopic parameters (positions of absorption and two fluorescence maxima, the ratio of their intensities and the fluorescence quantum yield) can be measured in this solvent range with multiparametric analysis of the data. In terms of solvent polarity, the shifts in both emission bands and their intensity ratio demonstrate a good correlation with empirical polarity scales E_T^N, P_y and SPP, while the absorption spectra reveal some deviations for the tested oxygen-containing solvent molecules. A good cross-correlation is observed between fluorescence spectral shifts and the ratio of band intensities. The latter provides the means for a dramatic amplification of solvent response. Thus, a new approach for ultrasensitive scaling and probing the solvent polarity in the low-polararity range can be suggested. It involves very simple ratiometric measurements at two emission bands and can be posed for a variety of applications. We present examples of these applications for distinguishing of polarities between methylated benzene derivatives, for quantitative assay of polar impurities in low-polar solvents and for detection of the changes of solvent polarity as a function of temperature.