New redox system: Trichloromethylarene —pyridine base

A redox reaction of trichloromethylarenes with pyridines results in respective N-(-chloroarylmethyl)substituted pyridiniurn chlorides which give, on hydrolysis, aromatic aidehydes and 4-chloropyridines or 1, 4-bipyridiniurn salts.N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 117913 Moscow, Russia. Published in Khimiya Geterotsiklicheskikh Soedinenii, No. 10, pp. 1373–1375, October, 1995. Original article submitted September 5, 1995.     

Amperometric urea biosensor based on urease and electropolymerized toluidine blue dye as a pH-sensitive redox probe

The electropolymerized toluidine blue film deposited on the glassy carbon electrode show amperometrically detectable pH sensitivity. This feature of polytoluidine blue (PTOB) film was used for a construction of an amperometric urea biosensor. We have observed a linear shift of the formal redox potential with increasing pH value between 4 and 8 giving the slope of 81 mV(Delta) pH(-1). Polytoluidine blue film has had a significantly increased stability and higher electrochemical activity compared to the adsorbed monomeric dye. The polytoluidine blue urea biosensor has been operating at a working potential of -200 mV vs. SCE. The sensitivity of the biosensor was 980 nA mM(-1) cm(-2). The biosensor showed linearity in concentration range up to 0.8 mM with the detection limit of 0.02 mM (S/N=3).     

Transfection mediated by gemini surfactants: engineered escape from the endosomal compartment

The structure of the lipoplex formed from DNA and the sugar-based cationic gemini surfactant 1, which exhibits excellent transfection efficiency, has been investigated in the pH range 8.8-3.0 utilizing small-angle X-ray scattering (SAXS) and cryo-electron microscopy (cryo-TEM). Uniquely, three well-defined morphologies of the lipoplex were observed upon gradual acidification: a lamellar phase, a condensed lamellar phase, and an inverted hexagonal (H(II)) columnar phase. Using molecular modeling, we link the observed lipoplex morphologies and physical behavior to specific structural features in the individual surfactant, illuminating key factors in future surfactant design, viz., a spacer of six methylene groups, the presence of two nitrogens that can be protonated in the physiological pH range, two unsaturated alkyl tails, and hydrophilic sugar headgroups. Assuming that the mechanism of transfection by synthetic cationic surfactants involves endocytosis, we contend that the efficacy of gemini surfactant 1 as a gene delivery vehicle can be explained by the unprecedented observation of a pH-induced formation of the inverted hexagonal phase of the lipoplex in the endosomal pH range. This change in morphology leads to destabilization of the endosome through fusion of the lipoplex with the endosomal wall, resulting in release of DNA into the cytoplasm.     

Identification (GC and GC-MS) of unsaturated acetates in Elasmopalpus lignosellus and their biological activity (GC-EAD and EAG)

Two insect colonies of Elasmopalpus lignosellus were reared in our laboratory, the first being initiated from pupae obtained from a cornfield in the region of Sete Lagoas, Minas Gerais and the second from a cornfield in the region of Goiania, Goiás. From the two colonies, two extracts were prepared from the pheromone glands of virgin E. lignosellus females. The extract obtained from the first colony was designated as extract 1 while the extract obtained from the second colony was designated as extract 2. Extract 1 was analyzed by gas chromatography-mass spectrometry (GC-MS) with (Z)-9-hexadecenyl acetate [(Z)-9-HDA] and (Z)-11-hexadecenyl acetate [(Z)-11-HDA] being identified and confirmed by the formation of DMDS derivatives. In addition, a third acetate, which could be either (E)-8-hexadecenyl acetate [(E)-8-HDA] or (E)-9-hexadecenyl acetate [(E)-9-HDA] was detected by GC-MS. Extract 2 was analyzed by gas chromatography (GC) and gas chromatography-electroannetography (GC-EAD) revealing the presence of (Z)-11-HDA and (Z)-9-TDA. In addition, the same compounds elicited a response with the E. lignosellus male antenna obtained from the second insect colony. Electroantennography (EAG) screening with the male E. lignosellus antenna (obtained from the second insect colony) was conducted with the 23 possible tetradecenyl acetates (TDA) and 22 hexadecenyl acetates (HDA) as standards. Out of the 23 TDA isomers evaluated, only (Z)-9-TDA elicited a response and out of the 22 HDA [(Z) and (E) isomers gamma2 to delta13] evaluated only (Z)-11-HDA elicited a response. The acetate compositions of two extracts obtained from insects originating from the two states (Minas Gerais and Goiás) of Brazil were different from one another as well as from that obtained from insects in Tifton, GA, USA. The bioactivity data (GC-EAD) of the extract 2 differed from those reported for the Tifton, GA, USA population. These data suggest polymorphism in relation to the insect populations found in Brazil and in the USA. The possibility of the existence of an E. lignosellus sub-species cannot be ruled out.     

Photoinduced axial ligation and deligation dynamics of nonplanar nickel dodecaarylporphyrins

The ground- and excited-state metal-ligand dynamics of nonplanar nickel(II) 2,3,5,7,8,10,12,13,15,17,18,20-dodecaphenylporphyrin (NiDPP) and two fluorinated analogues (NiF(20)DPP and NiF(28)DPP) have been investigated using static and time-resolved absorption spectroscopy in toluene and in ligating media that differ in basicity, aromaticity, and steric encumbrance. Because of the electronic and steric consequences of nonplanarity, NiDPP does not bind axial ligands in the ground state, but metal coordination does occur after photoexcitation with multistep dynamics that depend on the properties of the ligand. Following the structural relaxations that occur in all nickel porphyrins within approximately 10 ps, ligand binding to photoexcited NiDPP is progressively longer in pyridine, piperidine, and 3,5-lutidine (25-100 ps) but does not occur at all in 2,6-lutidine in which the ligating nitrogen is sterically encumbered. The transient intermediate that is formed, which nominally could be either a five- or six-coordinate species, also has a ligand-dependent lifetime (200-550 ps). Decay of this intermediate occurs partially via ligand release to re-form the uncoordinated species, in competition with binding of the second axial ligand and/or conformational/electronic relaxations (of a six-coordinate intermediate) to give the ground state of the bis-ligated photoproduct. The finding that the photoproduct channel principally depends on ligand characteristics along with the time-evolving spectra suggests that the transient intermediate may involve a five-coordinate species. In contrast to NiDPP, the fluorinated analogues NiF(20)DPP and NiF(28)DPP do coordinate axial ligands in the ground state but eject them after photoexcitation. Collectively, these results demonstrate the sensitivity with which the electronic and structural characteristics of the macrocycle, substituents, and solvent (ligands) can govern the photophysical and photochemical properties of nonplanar porphyrins and open new avenues for exploring photoinduced ligand association and dissociation behavior.     

Miscibility of zinc sulfide and zinc phosphide

Solid solutions in the system zinc sulfide/zinc phosphide (Zn(2+)(x)S(2-2xP(2x)) were investigated using the cyclic cluster model within the semiempirical MSINDO method. Results of cyclic cluster calculations for binding energies of the perfect ZnS and Zn(3)P(2) are presented and compared with the experimental data. The miscibility of ZnS and Zn(3)P(2) over the whole composition range of 0 < x < 1 was investigated by calculating the Gibbs free energy of mixing Delta(M)G for different values of x. A miscibility gap was found at both ends of the composition range and compared with experimental data.     

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     

Automatic flow procedure for the determination of glycerol in wine using enzymatic reaction and spectrophotometry

An automatic flow procedure for the determination of glycerol in wines by employing a flow system based on multicommutation and enzymatic reaction is described. Glycerol dehydrogenase was immobilized on aminopropyl glass beads and packed into a column that was coupled to the flow system. The NADH produced by the enzymatic reaction was monitored by spectrophotometry at 340 nm and its radiation absorption presented a relationship with glycerol concentration. The system manifold comprised a set of three-way solenoid valves controlled by a microcomputer, which was furnished with electronic interfaces and runs a software that was designed to carry out on-line sample dilution, reagent addition, and data acquisition. The procedure allows the determination of glycerol in wine samples without any prior pretreatment. The procedure presented as profitable features a linear response range between 2.0 and 10.0 g l⁻¹ glycerol (R=0.998), a detection limit of 0.006 g l⁻¹ glycerol, a relative standard deviation of 1.8% (n=14) for a typical wine sample presenting 5.3 g l⁻¹ glycerol, a sampling throughput of 33 determinations per hour, and a NAD⁺ consumption of 0.8 mg per determination. The results were compared with those obtained using a reference method and no significant difference at 90% confidence level was observed.     

NMR detection of intermolecular NH.OP hydrogen bonds between guanidinium protons and bisposphonate moieties in an artificial arginine receptor

Hydrogen bonding plays a major role in the selective recognition of guanidinium groups by receptor molecules. The present NMR investigation provides direct experimental evidence of hydrogen bonds in an artificial arginine receptor complex consisting of alpha-N-benzoylarginine ethyl ester and a bisphosphonate tweezers molecule. trans-Hydrogen bond 2hJHP couplings between the phosphonate moieties and individual guanidinium protons as well as the amide proton have been detected by [1H,31P]-HMBC and [31P,1H]-INEPT experiments. The detected hydrogen bonding network in the investigated artificial arginine receptor shows a symmetrical end-on interaction of the guanidinium moiety, which enables concerted rotations and deviates from the structure proposed for the biological arginine fork.     

Production of high melt strength polypropylene by gamma irradiation

High melt strength polypropylene (HMS-PP) has been recently developed and introduced in the market by the major international producers of polypropylene. Therefore, BRASKEM, the leading Brazilian PP producer, together with EMBRARAD, the leading Brazilian gamma irradiator, and the IPEN (Institute of Nuclear Energy and Research) worked to develop a national technology for the production of HMS-PP. One of the effective approaches to improve melt strength and extensibility is to add chain branches onto polypropylene backbone using gamma radiation. Branching and grafting result from the radical combinations during irradiation process. Crosslinking and main chain scission in the polymer structure are also obtained during this process. In this work, gamma irradiation technique was used to induce chemical changes in commercial polypropylene with two different monomers, Tri-allyl-isocyanurate (TAIC) and Tri-methylolpropane-trimethacrylate (TMPTMA), with concentration ranging from 1.5 to 5.0 mmol/100 g of polypropylene. These samples were irradiated with a ⁶⁰Co source at dose of 20 kGy. It used two different methods of HMS-PP processing. The crosslinking of modified polymers was studied by measuring gel content melt flow rate and rheological properties like melt strength and drawability. It was observed that the reaction method and the monomer type have influenced the properties. However, the concentration variation of monomer has no effect.