Subsequent Carboxymethylation Research Articles

Alkali-activation of cellulose nanofibrils to facilitate surface chemical modification under aqueous conditions

In this study, we developed a surface-activation technique for cellulose nanofibrils (CNFs) using mild-alkali and aqueous conditions. CNFs were initially processed using the aqueous counter collision (ACC) method to produce Janus-type amphiphilic CNFs with both hydrophilic and hydrophobic faces on the surface of a single nanofibril (ACC-CNF). Selective functionalization of the hydroxy groups on the hydrophilic faces creates an opportunity to develop novel nano-building blocks that introduce heterogeneous and tailored surface characteristics into the design of nanomaterials. In this study, alkaline conditions were used to activate the hydroxy groups on the surface of ACC-CNFs as a pre-treatment for the partial crystalline transformation from cellulose I to cellulose II. We found that alkali treatment with sodium hydroxide (NaOH) solutions (concentration range 1–7 wt%) did not fully transform the structure of ACC-CNFs into cellulose II, nor change the morphology of nanofibrils, as seen from their wide-angle X-ray diffraction patterns and atomic force microscopy images. We also found that the hydroxy groups at the surface region of the ACC-CNFs were sufficiently reactive under the moderate alkali and aqueous conditions to undergo subsequent carboxymethylation. Therefore, alkali treatment of ACC-CNFs with a 1–7 wt% NaOH solution rendered the surface of the ACC-CNFs as sufficiently reactive for chemical modification without morphological changes. This simple method for surface activation of CNFs can be useful in the development of future sustainable and novel materials for a variety of applications.

Redox-sensitive, PEG-shielded carboxymethyl PEI nanogels silencing MicroRNA-21, sensitizes resistant ovarian cancer cells to cisplatin

A series of branched polyethylenimine (PEI) modifications including PEGylation (PEG2k-PEI) for steric shielding, redox-sensitive crosslinking for synthesis PEG2k-PEI-ss nanogels and subsequent carboxymethylation (PEG2k-CMPEI-ss) for modulation of the polymer pka have been introduced for cellular delivery of Anti-miR-21. The synthesis was characterized using 1H NMR, FTIR, TNBS, potentiometric titration, particle size and ζ potential. Loading of Anti-miR-21 at various N/P ratios was investigated by gel retardation, ethidium bromide dye exclusion, heparin sulfate competition and DNase I digestion experiments. The miR-21 silencing was measured by stem-loop RT PCR in A2780 ovarian cancer cell lines whether it is sensitive to resistant to cisplatin. It has been shown that PEG2k-CMPEI-ss was well suited for delivery of Anti-miR-21 in terms of nucleic acid loading, preservation against extracellular matrix and nucleases and sequence-specific silencing of miRNA-21 in vitro. Moreover, it has been demonstrated that pre-treating cells with Anti-miR-21 loaded nanogels can sensitize them to cis-Pt even at non-toxic concentraions. The results indicate that PEG2k-CMPEI-ss mediated microRNA delivery can be considered as a novel strategy for ovarian cancer therapy.

Impact of pre-treatments on properties of lignocelluloses and their accessibility for a subsequent carboxymethylation

In this issue, different chemical (alkaline and sulfite pulping, ozonolysis) and mechanical (vibratory ball milling) pre-treatments were utilized for activating wheat straw and beech sawdust prior to carboxymethylation. Detailed analysis by a range of methods, including Klason-lignin, cellulose and hemicellulose quantification, Powder-X-ray diffraction (PXRD) and attenuated total reflection (ATR) IR spectroscopy, enabled the investigation of material alterations. Subsequently, carboxymethylation was carried out with both untreated and activated materials, allowing the evaluation of activation steps by determining degrees of substitution with carboxymethyl groups (DSCM). Moreover, carboxymethylation conditions were optimized, realizing high DSCM of up to 1.05. Results further revealed that ball milling enhanced the subsequent conversion; whereas chemical pre-treatments did not effectively increase material accessibilities. Further studies on chemically untreated materials emphasized that a highly reactive surface was already generated in the course of the carboxymethylation, inter alia through the concomitant dissolution of matrix components.

Composite films prepared from agricultural by-products

In our study we used holocelluloses from sugar beet and bagasse for film preparation. Films from sugar beet holocellulose have better mechanical properties than from bagasse holocellulose. By subsequent carboxymethylation of bagasse holocellulose, films with better properties were produced. Specimens prepared from combined sugar beet and bagasse carboxymethylated holocellulose had the best mechanical properties. The results could be explained by the ratios of cellulose, arabinan, polygalacturonan and xylan content in individual films, based on the elemental analysis data. The use of microwaves to prepare holocellulose film speed up the process, but negatively affected the mechanical properties. Lignin content of the sugar beet holocellulose and bagasse samples was low and did not affect the mechanical properties. Both types of agricultural by-products could be used for preparation of composite film with high strength and stiffness suitable for broad range of applications.

An Efficient Method for Preparing Carboxymethyl Chitosan by the Activation of Chitosan

To improve carboxymethylation reaction, chitosan as reactant was activated to change its morphology and compact structure. The crystallization properties of chitosan before and after activation were characterized by X-ray diffraction analysis. Different preparation methods were compared using activated, powdery or flake chitosan as reactants, respectively. And the influences of reaction time and temperature on the yield, the intrinsic viscosity and the degree of substitution of carboxymethyl chitosan were also investigated. We found that, the activation treatment changed the crystallization properties of chitosan, which greatly accelerated the subsequent carboxymethylation reaction; and higher yield of carboxymethyl chitosan with considerable intrinsic viscosity was obtained under the reaction conditions of a shorter time and a milder temperature.

Preparation of a New Polystyrene Supported-Ethylenediaminediacetic Acid Resin and its Sorption Behavior toward Divalent Metal Ions

A new polystyrene-supported ethylenediaminediacetic acid resin has been synthesized through a reaction between the amination of the commercially available 4-chloromethyl polystyrene polymer with ethylenediamine and the subsequent carboxymethylation with monobromoacetic acid, using ethylenediamine as spacer. The chelation behavior of this resin toward the divalent metal ions Cu2+, Ni2+, Zn2+, and Pb2+ in aqueous solutions was investigated. Batch equilibration experiments were carried out as a function of contact time, pH, amount of metal-ion, and polymer mass. The amount of metal-ion uptake of the polymer was determined by using atomic absorption spectrometry (AAS). Results of the investigation revealed that the resin exhibited higher capacities and a more pronounced adsorption toward Cu2+ and that the metal-ion uptake follows the order: Cu2+ > Zn2+ > Ni2+ > Pb2+. The adsorption and binding capacity of the resin toward the various metal ions investigated are discussed.

Preparation and characterization of micron‐sized non‐porous magnetic polymer microspheres with immobilized metal affinity ligands by modified suspension polymerization

AbstractA novel preparation method of micron‐sized non‐porous magnetic polymer microspheres with immobilized metal affinity ligands was developed. A modified suspension polymerization of methacrylate (MA) and divinylbenzene (DVB) was performed in the presence of oleic acid‐coated magnetic Fe3O4 nanoparticles to obtain magnetic poly (methacrylate‐divinylbenzene) (mPMA‐DVB) microspheres. Through ammonolysis using ethylenediamine (EDA) and subsequent carboxymethylation with chloroacetic acid, magnetic polymer microspheres with chelate ligands of iminodiacetic acid (IDA) were obtained. Charging with copper ions resulted in magnetic polymer microspheres capable of binding proteins that display metal affinity. The morphology, magnetic properties, and composition of magnetic polymer microspheres were characterized with scanning electron microscopy (SEM), vibrating sample magnetometer (VSM), and Fourier transform infrared spectroscopy (FTIR), respectively. Bovine hemoglobin (BHb) was adopted as a model protein to investigate their affinity adsorption capacity. It was found that the adsorption capacity was as high as 168.2 mg/g microspheres and with rather low non‐specific adsorption. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 96: 2174–2180, 2005

Synthesis and SAM formation of water soluble functional carboxymethylcelluloses: thiosulfates and thioethers

Functional thiomethyl and thiosulfate derivatives of carboxymethylcellulose (CMC, DS = 0.9) were synthesized by nucleophilic displacement reactions. Alkylation of CMC by allyl glycidyl ether took mainly place at the primary positions of the cellulose backbone and yielded a 6-O-(3′-allyloxy-2′-hydroxypropyl)-CMC 1 with a partial DS of 3′-allyloxy-2′-hydroxypropyl substituents DSallyl of up to 0.43. Addition of tetrathionate to the allyl groups gave rise to 6-O-(2′′,3′′-bis(thiosulfato)propoxy-2′-hydroxypropyl)-CMC 2. As the addition of tetrathionate was sluggish and incomplete, alternatively bromine was added and the resulting dibromide was substituted by thiosulfate. A 40% conversion of the allyl groups was achieved by this two-step procedure. On the other hand, the addition of bromine to 1 in aqueous solution almost quantitatively yielded the bromohydrin derivative which was converted by displacement reaction with thiosulfate to 6-O-(2′′-hydroxy-3′′-thiosulfatopropoxy-2′-hydroxypropyl)-CMC 4. 6-Thiomethyl-6-deoxy-CMC 6 was synthesized by displacement reaction of 6-O-tosylcellulose with sodium methylsulfide and subsequent carboxymethylation of the cellulose backbone. A partial DS of thiomethyl substituents DSThM=0.65 exclusively at the primary positions was obtained. All functional CMC derivatives, 2, 4, and 6 were readily available in gram quantities, rather stable and highly water soluble for pH > 3. On gold surfaces they form self-assembled monolayers (SAMs) with thicknesses of 1.2 to 2.4 nm as determined by surface plasmon resonance (SPR).

Starch Derivatives of High Degree of Functionalization. 4. Homogeneous Tritylation of Starch and Subsequent Carboxymethylation

The synthesis of monomethoxytriphenylmethyl (MMtrityl) starch and the subsequent carboxymethylation of the 6-O-functionalized products were investigated. The trityIation both in N,N-dimethylacetamide (DMA)/LiCl and in dimethyl sulfoxide (DMSO) occurred homogeneously. The highest degree of substitution of trityl groups (DSTrityI) obtained after a single conversion step was 0.77 in both solvents. A complete functionalization of primary OH-groups was achieved only with unsubstituted triphenylmethyl chloride in these reaction media. In case of monomethoxytriphenylmethyl chloride (MMTMC) as reagent an additional conversion step is necessary to synthesize products with a DSTrityI = 1. The structure of the products was analyzed by FTIR- and 13C-NMR spectroscopy. Subsequent carboxymethylation of the MMtrityl starch samples leads to products with a preferred functionalization of the unprotected secondary OH-groups. After removal of the trityl moieties, the DSCM and the distribution of carboxymethyl groups within the anhydroglucose unit was investigated by means of HPLC and 1H-NMR spectroscopy. The carboxymethylation was more effective at O-2 than at O-3. In case of ether products with DSTrityl < 1 a partial substitution of the primary OH group took place as well.

Starch Derivatives of High Degree of Functionalization. 4. Homogeneous Tritylation of Starch and Subsequent Carboxymethylation

The synthesis of monomethoxytriphenylmethyl (MMtrityl) starch and the subsequent carboxymethylation of the 6-O-functionalized products were investigated. The trityIation both in N,N-dimethylacetamide (DMA)/LiCl and in dimethyl sulfoxide (DMSO) occurred homogeneously. The highest degree of substitution of trityl groups (DSTrityI) obtained after a single conversion step was 0.77 in both solvents. A complete functionalization of primary OH-groups was achieved only with unsubstituted triphenylmethyl chloride in these reaction media. In case of monomethoxytriphenylmethyl chloride (MMTMC) as reagent an additional conversion step is necessary to synthesize products with a DSTrityI = 1. The structure of the products was analyzed by FTIR- and 13C-NMR spectroscopy. Subsequent carboxymethylation of the MMtrityl starch samples leads to products with a preferred functionalization of the unprotected secondary OH-groups. After removal of the trityl moieties, the DSCM and the distribution of carboxymethyl groups within the anhydroglucose unit was investigated by means of HPLC and 1H-NMR spectroscopy. The carboxymethylation was more effective at O-2 than at O-3. In case of ether products with DSTrityl < 1 a partial substitution of the primary OH group took place as well.

Synthesis and Carboxymethylation of Organo-Soluble Trifluoroacetates and Formates of Cellulose

Soluble cellulose intermediates are converted into carboxymethyl cellulose (CMC) with a high degree of substitution (DS CMC ) of up to 2.2 by removing the solubilizing ester groups in an one-step synthesis in dimethylsulfoxide (DMSO) with suspended solid NaOH powder as a base and monochloroacetate within 4 hours at 70°C. The reaction is a pseudohomogeneous conversion, i.e., it proceeds under temporary inhomogeneity of the substrate (cellulose). As confirmed by means of HPLC analysis, these CMCs contain a significantly higher amount of both 2,3,6-tri-O-carboxymethylated and unsubstituted units in the polymer chain than those obtained in a slurry of cellulose in isopropanol/water (totally heterogeneous conversion) at comparable DS CMC . In contrast to heterogeneously prepared samples, the CMCs show a distribution of carboxymethyl groups within the anhydroglucose unit in the order 0-6 > 0-3 > 0-2 as revealed by means of 13 C- and 1 H-NMR studies. As intermediates for subsequent carboxymethylation, organo-soluble cellulose trifluoroacetates (CTFA) and cellulose formates (CF) have been synthesized and studied. The CFs were obtained by a new, convenient synthesis method consisting of the acylation of cellulose with mixtures of formic acid and phosphorus oxychloride within 4 hours at room temperature. The CFs with a degree of substitution (DS CF ) of 2.2 are soluble in DMSO and DMF, and they show a complete substitution of the primary OH groups as confirmed by 13 C-NMR studies and HPLC analysis after permethylation and chain degradation. The CTFAs (DS CTFA = 1.5) prepared by acylation of cellulose with a mixture of trifluoroacetic acid/trifluoroacetic anhydride also show a preferred substitution of the C-6 position.

Detection of Metallothionein (MT) Proteins with Radiolabeled [14C]Iodoacetamide

Carboxymethylation of protein samples prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is known to improve the transfer and detection of metallothioneins (MTs) on nitrocellulose membranes by Western blotting. Dissociation of metals from MTs by denaturing the samples with SDS, reduction of sulfhydryl (-SH) residues on MT-associated cysteines with dithiothreitol (DTT), and subsequent carboxymethylation of the cysteine residues with iodoacetamide overcome disulfide bond formation between MT molecules, preventing their aggregation, and allow for the electrophoretic transfer and detection of MTs by Western blotting. In the present study, advantage is taken of cysteine residue bond formation by the addition of 14C-radiolabeled iodoacetamide to the carboxymethylation process to validate β-emission autoradiography as an alternative method to Western blotting. The analysis of MTs by β-emission autoradiography suggests that treatment of neonatal rat primary astrocyte cultures with cadmi...

Development of immobilized metal affinity chromatography : I. Comparison of two iminodiacetate gels

Two iminodiacetate (IDA) gels were prepared by different synthetic routes: (a) direct coupling of IDA to epoxy-activated Sephadex G-25; and (b) a two-step synthesis involving initial introduction of amino groups into epoxy-activated Sephadex G-25 and their subsequent carboxymethylation. The efficiency of coupling of IDA to the epoxy-activated matrix was studied in detail as a function of pH in the range from 8.0 to 12.5. The IDA gel produced by the direct coupling procedure at the optimum pH of 10.5 was found to retain ca. 450 μmol of Cu 2+ per g dry weight. Different α-haloacids were explored as carboxymethylating agents for the second step of the two-step preparation. IDA gels produced with the use of bromoacetic acid were found to retain ca. 370 μmol of Cu 2+ per g dry weight. A novel chelating gel having a “hybrid” carboxyl/sulphonic ligand was also synthesized and its chelating properties were compared to the standard IDA gels. This gel retained only 180 μmol of Cu 2+ per g dry weight. The retention of Cu 2+ in the presence of the competing ligand, glycine, was inferior to that obtained with the IDA ligand.

Carboxymethylation of thiol groups in ovalbumin: implications for proteins that contain both thiol and disulfide groups.

The cysteine residues of hen ovalbumin were S-carboxymethylated with non-radioactive iodoacetic acid under various conditions by altering the pH at which the protein was denatured in 8 M urea, by using different molar ratios of non-radioactive iodoacetic acid to cysteine and by varying the time at which carboxymethylation was commenced after denaturing conditions had been applied. Under the various conditions, the thiol groups were carboxymethylated to different extents, the residual thiol groups being measured by reaction with 5,5'-dithiobis(2-nitrobenzoic acid) in the presence of sodium dodecyl sulfate. When ovalbumin is carboxymethylated in alkaline urea, it unfolds slowly and the carboxymethylation is incomplete even with 150-fold excess iodoacetic acid. The known rapid thiol-disulfide exchange that occurs at alkaline pH values makes this method of carboxymethylation unsuitable as a preliminary step for blocking the native cysteine residues of ovalbumin before reduction and labelling the thiol groups formed by reduction of the disulfide bonds. Titration of the thiol groups of ovalbumin in 6 M guanidine hydrochloride or 1% (w/v) sodium dodecyl sulfate at pH 8.2 with 5,5'-dithiobis(2-nitrobenzoic acid) is more rapid than in 8 M urea and these solvents would be preferable for studies of the disulfide-bonded sequences. Denaturation of ovalbumin in acidic 8 M urea is a very rapid process, and under mild acid conditions thiol-disulfide interchange is much slower. Subsequent carboxymethylation of the cysteine residues at alkaline pH with 150-fold excess iodoacetic acid results in complete carboxymethylation and the carboxymethylated ovalbumin can be reduced and labelled with radioactive iodoacetic acid with specific labelling of the half-cystine residues involved in the disulfide bond. The results are discussed in relation to the allocation of half-cystine residues in other protein systems that contain both thiol and disulfide groups.

Studies on the role of sulfhydryls in the myosin ATPase. Characterization of the site of modification by the bifunctional sulfhydryl reagent p-N,N'-phenylenedimaleimide.

Myosin has been modified with near stoichiometric amounts of the bifunctional reagent [14C]p-N,N'-phenylenedimaleimide (pPDM) in the presence of MgADP under conditions which abolish its ATPase activity. Subsequent carboxymethylation and CNBr cleavage results in the 14C label being associated with a single polypeptide of Mr approximately 10,000. Amino acid composition and partial sequence analysis of this peptide showed that it corresponded to the peptide containing -SH1 and -SH2 sequenced by Elzinga and Collins (Elzinga, M., and Collins, J.H. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 4281-4284) and to the peptide labeled at -SH1 or -SH2 by N-ethylmaleimide by Kunz et al. (Kunz, P.A., Walser, J.T., Watterson, J.G., and Schaub, M.C. (1977) FEBS Lett. 83, 137-140). These data indicating that pPDM does label the -SH1- and -SH2-containing region in myosin by covalently bridging them and shows that in the presence of MgADP these thiols can approach to within 12 to 14 A.

Effect of chemical modification and fragmentation on antigenic determinants of internal protein p30 and surface glycoprotein gp70 of type C retroviruses.

The effects of protein modification on the antigenic determinants of p30 and gp70 of type C retroviruses were investigated by using solid-phase competition radioimmunoassays. Proteins were modified by reduction with 2-mercaptoethanol and subsequent carboxymethylation of SH groups with iodoacetamide or by amidination of alpha and epsilon amino groups with methylacetimidate. The type-specific determinants of gp70 were found to be conformational in nature, as they were destroyed by these chemical modifications. Group- and interspecies-specific determinants of gp70 antigens, however, appear to be sequential and do not involve residues susceptible to these chemical reagents. Conformation-dependent type-specific determinants of p30 were affected only by methylacetimidate. Group- and interspecies-specific determinants of p30 are similar to those of gp70 in that they also appear to be sequential antigenic sites. Therefore, the broadly reactive group- and interspecies-specific determinants of gp70 and p30 can be followed into small peptides. Accordingly, a cyanogen bromide cleavage fragment derived from the carboxyl-terminal one-third of Rauscher leukemia virus p30 was found to carry group-specific determinants but no detectable interspecies-specific determinants. In contrast, a peptide obtained by limited trypsin cleavage of p30, which was derived from the NH(2)-terminal region of the protein, contained at least one of the interspecies determinants shared with feline leukemia virus p27.

The Use of Soybean Trypsin Inhibitors as Phosphorylatable Substrates for a Rat Liver Protein Kinase

Phosphorylation by a cAMP-independent rat liver protein kinase of protein substrates containing the structural feature required by mammary gland casein kinase (-Ser-X-Glu/Asp) has been demonstrated. In particular, the Bowman-Birk Soybean trypsin inhibitor, which is characterized, like other legume protease inhibitors, by clusters of acidic residues near the C-terminal side of seryl residue(s), proved to be a good model substrate for the protein kinase. Its phosphorylation, involving the Ser 65 residue, is apparently hindered by the binding of trypsin, while it is stimulated by unfolding induced by reduction and subsequent carboxy-methylation.

Hydrophobic fluorescent group coupled to Taka-amylase A. Change of its environmnt accompanying splitting of disulfide bonds.

AbstractNative Taka‐amylase A (α‐amylase produced by Asp. oryzae) was coupled with 1‐dimethylaminonaphthalene‐5‐sulfonyl chloride. When disulfide bonds of the modified enxyme were split by reduction or by reduction and subsequent carboxymethylation, its fluorescent properties changed markedly. It was suggested that the hydrophobic dye group is incorporated into a hydrophobic region as the consequence of the flexibility gained by the polypeptide chain by the cleavage of disulfide bonds.

Alkylation of cytochromes c. I. Properties of alkylated beef cytochrome c.

Beef heart cytochrome c was alkylated at methionine-65 and methionine-80 at pH 3. The alkylation caused the complete loss of the oxidase activity with cytochrome a as well as drastic changes in other properties. The alkylation reaction was faster with mono-iodoacetic acid than with its amide at pH 3. At pH 5–6, the reaction with mono-iodoacetic acid occurred more slowly than at pH 3. Carboxymethylation occurred first at methionine-65 and histidine-33 without any appreciable loss of the enzymic activity. Subsequent carboxymethylation of methionine-80 seemed to cause the loss of the activity. Amino acid analyses and peptide maps of the reaction products supported the conclusions mentioned above and the conformational aspect of cytochrome c is discussed in the light of these results.