Diallyltartardiamide Research Articles

Imine bonding self-repair hydrogels after periodate-triggered breakage of their cross-links

The development of new materials with smart properties is currently expanding the development of new technologies. Therefore, the design of materials with novel sensitivities and smart behavior is important for the development of smart systems with automated responsivity. We have recently reported the synthesis of hydrogels, cross-linked by N,N'-diallyltartardiamide (DAT). The covalent DAT-crosslinking points have vicinal diols which can be easily cleaved with periodate, generating changes in the hydrogel properties, as well as generating valuable α-oxo-aldehyde functional groups useful for further chemical modification. Based on those findings, we envisioned that a self-healable hydrogel could be obtained by incorporation of primary amino functional groups, from 2-aminoethyl methacrylate hydrochloride (AEMA), coexisting with DAT into the same network. Herein, α-oxo-aldehyde groups generated after the reaction with periodate would arise in the immediate environment of amine groups to form imine cross-links. For this purpose, DAT-crosslinked hydrogels were synthesized and carefully characterized. The cleavage of DAT-crosslinks with periodate promoted changes in the mechanical and swelling properties of the materials. As expected, a self-healing behavior was observed, based on the spontaneous formation of imine covalent bonds. In addition, we surprisingly found a combination of fast vicinal diols cleavage and a low speed self-crosslinking reaction by imine formation. Consequently, it was found a time-window in which a periodate-treated polymer was obtained in a transient liquid state, which can be exploited to choose the final shape of the material, before automated gelling. The singular properties attained on these hydrogels could be useful for developing sensors, actuators, among other smart systems.

The use of N,N′-diallylaldardiamides as cross-linkers in xylan derivatives-based hydrogels

N,N′ -Diallylaldardiamides (DA) were synthesized from galactaric, xylaric, and arabinaric acids, and used as cross-linkers together with xylan (X) derivatives to create new bio-based hydrogels. Birch pulp extracted xylan was derivatized to different degrees of substitution of 1-allyloxy-2-hydroxy-propyl (A) groups combined with 1-butyloxy-2-hydroxy-propyl (B) and/or hydroxypropyl (HP) groups. The hydrogels were prepared in water solution by UV induced free-radical cross-linking polymerization of derivatized xylan polymers without DA cross-linker (xylan derivative hydrogel) or in the presence of 1 or 5 wt % of DA cross-linker (DA hydrogel). Commercially available cross-linker (+)- N,N′-diallyltartardiamide (DAT) was also used. The degree of substitution (DS) of A, B, and HP groups in xylan derivatives was analyzed according to 1H NMR spectra. The DS values for the cross-linkable A groups of the derivatized xylans were 0.4 (HPX-A), 0.2 (HPX-BA), and 0.4 (X-BA). The hydrogels were examined with FT-IR and elemental analysis which proved the cross-linking successful. Water absorption of the hydrogels was examined in deionized water. Swelling degrees up to 350% were observed. The swollen morphology of the hydrogels was assessed by scanning electron microscopy (SEM). The presence of cross-linkers in DA hydrogels had only a small impact on the water absorbency when compared to xylan derivative hydrogels but a more uniform pore structure was achieved.

Single step synthesis of carbohydrate monolithic capillary columns for affinity chromatography of lectins

Carbohydrate monolithic beds were synthesized in a single step in capillary columns to study affinity chromatography of lectins. In this method, carbohydrates (beta-galactose, beta-glucose, and alpha-mannose) with an easy to synthesize alkene terminated tetraethylene glycol spacer were used as functional monomers along the monomer 2-hydroxyethyl methacrylate (HEMA). As crosslinkers (+)-N,N'-diallyltartardiamide (DATD) and piperazine diacrylamide (PDA, 1,4-bisacryloyl-piperazine) were used. SEM showed the successful formation of monolithic beds in the capillary columns. The permeability of the columns was high. The specific interaction of the lectins Con A, Lens culinaris (LCA) and Arachis hypogaea (PNA) with the carbohydrate stationary phase was studied by frontal affinity chromatography (FAC). Con A and LCA were successfully eluted from the column using 0.1 M methyl-alpha-mannopyranoside and PNA with 0.1 M beta-galactose. Dissociation constants (Kd) for carbohydrate-lectin interactions were determined and compared with literature.

Preparation of a monolithic capillary column with immobilized α-mannose for affinity chromatography of lectins

A simple method for the preparation of an affinity monolithic (also called continuous bed) capillary column for α-mannose-specific lectins is described. 2-Hydroxyethyl methacrylate in combination with (+)- N, N´-diallyltartardiamide (DATD) and piperazine diacrylamide (PDA, 1,4-bisacryloyl-piperazine) as crosslinkers, were used as monomers for the monolith. After oxidation of DATD with periodate, α-mannose with spacer was bound to the aldehyde groups of the polymeric skeleton via reductive amination to form an affinity column for the separation, enrichment or binding studies of mannose-specific lectins. The permeability of the column was excellent. The porosity of the monolith was investigated by scanning electron microscope (SEM) and inverse size exclusion chromatography (ISEC). The affinity of the monolith was evaluated by frontal analysis (FA) and fluorescence microscopy (FM) using fluorescently labeled concanavalin (Con A). Frontal affinity chromatography showed a specific interaction of two different lectins with the α-mannose-modified monolith. According to FM the affinity sites were evenly distributed over the monolithic bed.

Isolation and partial characterization of a high molecular weight anionic glycoconjugate from transforming growth factor-beta treated bovine articular chondrocyte cultures.

A high molecular weight anionic glycoconjugate was isolated from the media of the transforming growth factor-beta treated chondrocyte cultures by anion-exchange chromatography on DEAE-Sephacel and Mono Q columns and was partially characterized. This high molecular weight anionic glycoconjugate was not detected in the non-treated (control) cultures. Characterization studies showed that the glycoconjugate is a non-reducible, non-collagenous glycoprotein containing O-linked, N-linked, and sialic acid substituted carbohydrate units. The isolated glycoconjugate stained "blue" with Stains All and migrated as a single band on sodium dodecyl sulfate gradient gels (2.5-10% acrylamide - diallyl tartardiamide) at an estimated molecular weight of 540 000. Amino acid and amino sugar analyses showed that it is rich in aspartic acid--asparagine, glutamic acid--glutamine, alanine, proline, and glycine, and contains galactosamine and glucoasmine. This transforming growth factor-beta inducible glycoprotein may be involved in cell differentiation and in the cartilage repair process. It may also be used as a marker to localize the biological activity of transforming growth factor-beta in articular cartilage.

Investigation of the properties of acrylamide bifunctional monomers (cross-linkers) by capillary zone electrophoresis

A series of cross-linkers [N,N′-methylenebisacrylamide (Bis), N,N′-(1,2-dihydroxyethylene)bisacrylamide (DHEBA), N,N′-diallyl- tartardiamide (DATD) and N,N′-bisacrylylcystamine (BAC)] were investigated as potential candidates of a novel class of polyacrylamide matrices, exhibiting high hydrophilicity, high resistance to hydrolysis and larger pore sizes than conventional polyacrylamide gels. The most hydrophilic cross-linker (DATD) exhibited first-order degradation kinetics in 0.1 M NaOH, suggesting that such a structure is electronically unstable, whereas the other cross-linkers displayed first-order kinetics. In addition, when used in highly cross-linked gels (for increasing pore size), DATD acts as an inhibitor of gel polymerization. Another interesting finding is an inverse relationship between the partition coefficient of such cross-linkers and the incorporation efficiency; the more hydrophobic members of the family exhibit a reduced conversion from monomer into polymer, which is more pronounced in highly cross-linked gels. When measuring the partition coefficient, DATD and DHEBA appear to be highly hydrophilic whereas BAC, owing to the two sulphur atoms in the molecule, appears to be extremely hydrophobic. The use of BAC in gels for protein separations should therefore be avoided.

Properties of acrylamide gels cross-linked with low concentrations of N, N′-diallyltartardiamide

The behavior of proteins on acrylamide gels cross-linked with low concentrations of N, N'-diallyltar-tardiamide (DATD) has been studied from a theoretical and practical point of view. Modifications in methodology and a technique for calculation of apparent molecular weights (MWs) allowed accurate determinations on gels from 7 up to 15% acrylamide. Comparison of viral protein patterns obtained with DATD- or N, N'-methylenebisacrylamide-cross-linked gels indicated greater resolution by DATD gels, particularly in the higher MW range. The polymerization reaction was studied kinetically and found to be efficient so long as the ratio of DATD to acrylamide was low. High resolution of DATD gels was attributed to the spontaneous formation of a gradient over much of the gel that retarded diffusion of the protein bands.

Polymerization kinetics of polyacrylamide gels I. Effect of different cross‐linkers

AbstractPolymerization kinetics of polyacrylamide gels, cross‐linked with the following: N,N′‐methylenebisacrylamide (Bis), N,N′‐bisacrylylcystamine (BAC), N,N′‐ diallyltartardiamide (DATD), N,N′(1,2‐dihydroxyethylene) bisacrylamide (DHEBA) and ethylene diacrylate (EDA), have been studied. The gels are polymerized directly in a spectrophotometer quartz cuvette and the kinetics followed at 283 nm (disappearance of the double bond) or at 600 nm (Tyndall effect due to turbidity of Bis, DHEBA and BAC gels). The order of reactivity of the various cross‐links appears to be: Bis ≅ DHEBA > EDA ≅ BAC ≫ DATD. The last cross‐link, (DATD), was found to actually be an inhibitor of gel polymerization, leading to highly unpolymerized gels, especially at high %C values. Bis and DHEBA gels, at 3 to 5 %C levels, are fully polymerized within 30 min at room temperature, while EDA and BAC gels, in the same %C range, require at least 3h. All cross‐links, when used above 10 %C, display quite slow polymerization kinetics, requiring overnight reaction for good conversion of monomers into polymer chains. When polymerizing liquid linear polyacrylamide (without cross‐link), an initial concentration of > 5% should be used and the reaction continued overnight, at room temperature or higher, for acceptable polymerization efficiency. On the basis of these data, a new structural model for highly cross‐linked gels is proposed.

Quantitative measurement of protein mass and radioactivity in N,N′-diallyltartardiamide crosslinked polyacrylamide slab gels

A simple technique for quantitative measurement of protein mass and radioactivity in samples separated by polyacrylamide slab gel electrophoresis is presented. The method is based on the sobubility in periodic acid of polyacrylamide gels crosslinked with N,N′-diallyltartardiamide (DATD). Proteins are first stained with Coomassie brilliant blue R-250, and the quantity of dye in solubilized gel sections is measured spectrophotometrically at 560 nm. Color stability is maximum at pH 3.0 and 25°C. Protein quantification is linear within most normal working conditions, and absorbance is not substantially affected by large variations in bandwidth. In addition, the radioactivity in gel slices from radioactive protein bands can be determined by liquid scintillation spectrometry following absorbance measurement. Neither periodic acid nor Coomassie brilliant blue R-250 cause substantial quenching in radioactive samples. Relative changes in synthesis and accumulation of myosin heavy chains in cultured skeletal muscle cells is presented to illustrate the technique.

Titration curves of proteins by combined isoelectric focusing-electrophoresis in highly porous polyacrylamide matrices

Highly cross-linked (highly porous) polyacrylamide gels as described in the literature were found to be poorly characterized support media. Diallyltartardiamide (DATD) cross-linked gels contain up to 80–90% unpolymerized DATD, which reacts with proteins and produces gluey and highly stretchable matrices. The conversion into polymer could not be affected by time or higher temperatures, since DATD is an inhibitor of polymerization, but only by adding very high amounts of persulphate. Increased levels of riboflavin actually decreased the polymerization efficiency. Highly cross-linked N,N′-methylenebisacrylamide (BIS) gels, at 40–50% C, are too hydrophobic and produce a collapsed matrix which keeps exuding water. A happy compromise are 30% C BIS gels, which are stable and allow unhindered migration of globular proteins up to 5·10 5 daltons. BIS and DATD gels can be stabilized by covalent binding to a glass surface precoated with Silane A-174. The order of reactivity for monomers appears to be: acrylamide > BIS ⪢ DATD. Polymerization conditions are described which allow better than 96% conversion of monomers into the polymer.

A method for the quantitative determination of protein incorporated in solubilizable polyacrylamide gels

Dense and light polyacrylamide gels containing N,N′-(1,2-dihydroxyethylene)bisacrylamide (DHEBA) or N,N′-diallyltartardiamide (DATD) as the crosslinker have been tested for their solubilization properties. Both types of gel can be dissolved in 10 m m periodic acid. The time and temperature required for complete dissolution of slabs of DHEBA crosslinked gels (12 h at 50°C), however, greatly exceed those required for dissolving slabs of DATD-polyacrylamide gel (0.5 h at 22°C). Bovine serum albumin kept under the respective dissolving conditions gave a lower response in the Lowry protein assay in instances where hot periodic acid had been used. Nearly independent of the type and concentration of their constituents, the different dissolved polyacrylamide gels interfere slightly with the Lowry assay by causing some “aspecific” color development. A method is outlined enabling a reliable quantitative determination of protein incorporated in DHEBA or DATD crosslinked polyacrylamide gels.

Properties of SDS—polyacrylamide gels highly cross-linked with N,N′-diallyltartardiamide and the rapid isolation of macromolecules from the gel matrix

Polyacrylamide gels cross-linked with N,N′-diallyltartardiamide (DATD) are, in contrast to gels cross-linked with N,N′-methylenebisacrylamide (Bis), readily solubilized by periodic acid (Anker, H. S., 1970, FEBS Lett. 7, 293), thus permitting efficient analyses of electrophoretically separated, labeled biological material. The capacities of polyacrylamide gels, cross-linked with Bis and DATD, to serve as media for electrophoretic separation of proteins, were compared. As DATD-cross-linked gels were inferior to equimolar Bis-crosslinked gels with 5% cross-linking ( C Bis = 5%) by the criteria of more pronounced swelling, markedly softer gels and, less concentrated and bended protein zones on electrophoresis and subsequent staining, gels cross-linked with different percentage CDATD were examined. The water regain of DATD-cross-linked gels, the retardation coefficients, and free mobilities of different proteins in equimolar Bis- and DATD-cross-linked gels were determined. When the DATD concentration in gels was increased to C DATD = 27%, gels assumed physical characteristics comparable to those cross-linked with Bis at C Bis = 5%. We report further the rapid, facile isolation of protein bands out of the gel matrix cross-linked with DATD. However, the isolation procedure results in an irreversible loss of biological activity.

A highly crosslinked, transparent polyacrylamide gel with improved mechanical stability for use in isoelectric focusing and isotachophoresis

Polyacrylamide gels highly (10–50%) crosslinked with N,N′-methylenebisacrylamide (BIS) provide large pore sizes but are mechanically labile. By substituting N,N′-diallyltartardiamide (DATD) for BIS, mechanical stability and firm adherence to glass walls is conferred to these gels, with only a minor decrease in pore size compared to highly BIS-crosslinked gels. The highly DATD-crosslinked gels have the further advantage of being transparent. It appears that highly DATD-crosslinked gels combining large pore size with mechanical stability are superior to presently used gel types in isoelectric focusing on polyacrylamide gel and isotachophoresis on polyacrylamide gel.