Comprehensive Synthetic Approach Research Articles

Geometric Transformation of Modified Multiwalled Carbon Nanotubes-Based Heterometallic Nanostructured Material: A Model for the Electrochemical Discrimination of Insecticides.

Multifunctional carbon-based materials exhibit a large number of unprecedented active sites via an electron transfer process and act as a desired platform for exploring high-performance electroactive material. Herein, we exemplify the holistic design of a heterometallic nanostructured material (MWCNTs@KR-6/Mn/Sn/Pb) formed by the integration of metals (Mn2+, Sn2+, and Pb2+) and a dipodal ligand (KR-6) at the surface of multiwalled carbon nanotubes (MWCNTs). First, MWCNTs@KR-6 was readily synthesized via a noncovalent approach, which was further sequentially doped by Mn2+, Sn2+, and Pb2+ to give MWCNTs@KR-6/Mn/Sn/Pb. The designed material showed excellent electrochemical activity for the discrimination of insecticides belonging to structurally different classes. In contrast to that of the individual building components, both the stability and electrochemical activity of heterometallic nanostructured material were remarkably enhanced, resulting in a magnificent electrochemical performance of the developed material. Hence, the current work reports a comprehensive synthetic approach for MWCNTs@KR-6/Mn/Sn/Pb synthesis by synergizing unique properties of the heterometallic complex with MWCNTs. This work also offers a new insight into the design of multifunctional carbon-based materials for discrimination of different analytes on the basis of their redox potential.

Competitive inhibition ELISA for the s-triazine herbicides: assay optimization and antibody characterization

The present work describes screening and selection of rabbit antisera raised against the s-triazine herbicides, characterization of antibody cross-reactivity and specificity, and improvements in ELISA sensitivity. These results stem from a comprehensive synthetic approach described previously for the production of s-triazine haptens. Both homologous and heterologous ELISA systems were examined by using heterology based on hapten conjugation position, spacer length, and/or alkyl substitution. Sensitivity to the target analyte and to nonspecific effects was least for homologous systems. Except for extreme differences in spacer length, heterology based on conjugation position provided the largest improvement in sensitivity to the target analyte. Matrix and solvent effects depended on the ELISA system used. Specificity studies using several antibodies in competitive inhibition ELISA with over 30 inhibitors showed that conjugation position and alkyl substitutions were important determinants of antibody specificity and that the antibodies recognized the immunizing hapten better than all other inhibitors tested. The resulting assays were capable of detecting the parent and related s-triazines a t low ppb to sub-ppb levels.

Inhibition of human choriotropin binding to receptor by human choriotropin alpha peptides. A comprehensive synthetic approach.

Synthetic overlapping peptides of the alpha-subunit of human chorionic gonadotropin (hCG) were made by solid-phase peptide synthesis employing a comprehensive synthetic approach. The entire primary structure of the alpha-subunit was synthesized as a series of nine consecutive peptides, each 15 residues in length, and overlapping with its two adjacent neighbors by 5 residues on each side. Receptor binding activity of each synthetic peptide was measured by the inhibition of binding of 125I-labeled hCG to rat ovarian receptor. Peptides alpha 21-35, alpha 31-45, alpha 71-85, and alpha 81-92 were shown to compete for binding with native hCG, thus demonstrating that at least two regions on the alpha-subunit may be part of the binding site(s) of the hormone. The low affinity of the peptides (10(-5)-10(-6) M) compared to native hormone (10(-10) M) for receptor is not unexpected due to the probability of discontinuous and multiple sites involved in receptor binding. An ultrapure preparation of hCG alpha-subunit also had low affinity (10(-5), suggesting that conformational changes upon combination with beta-subunit to form dimer or changes in conformation after binding are necessary for high affinity interaction. These results correlate with previous predictions of binding sites based on studies employing chemical and enzymatic modifications of intact hormone and show that synthetic peptide strategies are helpful in the elucidation of protein structure and function.

The regions of T-cell recognition on the extracellular part of the alpha chain of Torpedo californica acetylcholine receptor.

A comprehensive synthetic approach was employed to identify the continuous regions of T-cell recognition on the alpha-chain of Torpedo californica acetylcholine receptor (AChR). Eighteen synthetic consecutive overlapping peptides, of uniform size and overlaps, that spanned the entire extracellular part (residues 1-210) of the alpha chain were examined for their in vitro stimulation of lymph node cells from AChR-primed C57BL/6 (H-2b), C3H/He (H-2k), SWR(H-2q) and SJL (H-2s) mice. The T-cell recognition sites (T sites) in the AChR-primed mouse strains resided within six regions on the extracellular part of the alpha-chain. Three of the regions recognized by T cells coincided with regions recognized by antibodies (i.e. B cells) and one of these three regions also coincided with an alpha-neurotoxin binding region. It is noteworthy that, in addition to sites recognized by both T and B cells, the protein has at least two sites which are recognized exclusively by T cells and to which no detectable antibody responses are directed.

Immune recognition of human major histocompatibility antigens: localization by a comprehensive synthetic strategy of the continuous antigenic sites in the first domain of HLA‐DR2 β chain

A comprehensive synthetic approach, previously developed in this laboratory, has been applied to screen the entire first domain (residues 1-95) of the HLA-DR2 beta chain for the full profile of continuous regions that are recognized by human allo-antisera and anti-DR or anti-beta chain antisera raised in other species. Nine consecutive peptides, that were 15 residues each and overlapped by 5 residues, covering the first domain of the DR2 beta chain were synthesized, purified and characterized. The antibody-binding activities of the peptides were determined by radioimmunosorbent titrations. This established the full profile of peptides having specific antibody-binding activity with these various antisera. Three continuous antigenic sites were localized in this domain by all of antisera tested. Peptide 81-95 appeared to be the most immunodominant (i.e. bound the highest amounts of antibodies) in most antisera tested. Although their immunodominance varied with the antisera and boundary shifts were present, the submolecular regions recognized on the first domain of DR2 beta appeared to be similar with antisera against the DR2 (alpha beta I) complex or against the isolated beta chain. Furthermore, recognition was independent of the host species from which the antisera were obtained.

Antigenic structure of human haemoglobin. Localization of the antigenic sites of the β-chain in three host species by synthetic overlapping peptides representing the entire chain

A comprehensive synthetic approach is applied here to localize the continuous antigenic sites of the beta-chain of haemoglobin. The approach was based on the synthesis and purification of the following consecutive 15-residue peptides (each overlapping by five residues at both ends with the peptides preceding it and following it in the sequence): 1-15, 11-25 etc. Quantitative radiometric titrations of protein and peptide adsorbents were performed with 125I-labelled anti-haemoglobin antibodies from three different host species. The specificity of antibody binding to peptide adsorbents was confirmed by inhibition studies and by the binding specificity of antibodies isolated from peptide adsorbents. These studies established the full profile of antigenic beta-chain regions, which was found to be independent of the host species. Five major antigenic sites were localized, and their three-dimensional and structural characteristics are discussed in relation to the immune recognition of haemoglobin and other proteins.

Antibody recognition of ragweed allergen Ra3: Localization of the full profile of the continuous antigenic sites by synthetic overlapping peptides representing the entire protein chain

A comprehensive synthetic approach, for the localization of the full profile of the continuous antigenic sites on proteins, previously introduced by this laboratory, was applied here to localize the continuous antigenic sites of ragweed allergen, Ra3. The following 10 consecutive peptides, each comprising 15 residues (except for peptide 91-101) and overlapping each of its neighbors by 5 residues, were synthesized and purified: 1-15, 11-25, 21-35, 31-45, 41-55, 51-65, 61-75, 71-85, 81-95 and 91-101. Quantitative radiometric titrations of protein and peptide adsorbents were performed with 125I-labeled anti-Ra3 IgG antibodies from rabbit, outbred mouse and human antisera. The specificity of antibody binding to peptide adsorbents was confirmed by inhibition experiments. These studies established the full profile of antigenic (IgG-binding) sites of Ra3 and permitted comparison with the allergenic (IgE-binding) sites recently localized. It was found that the recognition by IgG antibodies was independent of the host species in which the antibodies were raised. Furthermore, the regions recognized by human IgE antibodies coincided with those recognized by IgG antibodies in three different species. Thus, Ra3 was found to have 4 continuous antigenic sites which occupy the same locations as the allergenic sites.

Localization of the continuous allergenic sites of ragweed allergen Ra3 by a comprehensive synthetic strategy

A comprehensive synthetic approach, previously introduced by this laboratory for the localization of the full profile of the continuous antigenic sites on proteins, was applied here to localize the continuous sites of ragweed allergen, Ra3, that are recognized by human anti-Ra3 IgE antibodies. The following 10 uniform and overlapping peptides were synthesized and purified: 1–15, 11–25, 21–35, 31–45, 41–55, 51–65, 61–75, 71–85, 81–95 and 91–101. Quantitative radiometric titrations of protein and peptide adsorbents with human IgE, established the full profile of allergenic (IgE binding) sites on Ra3. It was found that Ra3 has four continuous allergenic sites. Antibodies prepared against the IgE binding peptides bound to native Ra3. The findings are briefly discussed in relation to other protein antigenic structures and in terms of design of vaccines using synthetic sites.

Antigenic structure of human hemoglobin: Delineation of the antigenic site (site 2) within region 41–65 of the alpha chain by immunochemistry of synthetic peptides

A comprehensive synthetic approach consisting of a series of consecutive, uniform overlapping peptides encompassing the entire protein chain was recently used to determine the full antigenic profile of the α-chain of human hemoglobin (Hb). The peptides synthesized enabled the localization of five major “continuous” antigenic regions within the α chain. The present findings describe the delineation of an antigenic site (site 2) residing within the region 41–65. Ten peptides representing the α-chain regions 41–55, 51–65, 45–54, 45–56, 45–58, 45–60, 48–56, 49–56, 50–56, and 51–56 were synthesized and purified. Quantitative radioimmunoadsorbent titrations were used to determine binding to peptide adsorbents of radioiodinated anti-Hb antibodies that were raised in rabbit, goat, and outbred mouse. In one set of peptides, the N-terminal was fixed while the C-terminal end was increased by increments of two residues from Gln-54 to Lys-60 (i.e., peptides 45–54, 45–45, 45–58, and 45–60). Binding studies revealed that maximum antibody activity resided in peptide 45–45, indicating that Lys-56 marks the C-terminal boundary of the site. In the second set of peptides, the C-terminal was fixed at Lys-56 while the peptides were elongated at their N-terminal by one-residue increments from Gly-51 to Leu-48. Antibody-binding studies with these peptides indicated that Ser-49 defines the N-terminal boundary of the site. Therefore, the antigenic site within region 41–65 of the α chain comprises residues 49–56. The relevance of these findings to the immune recognition of Hb and other proteins is discussed.

A novel approach for localization of the continuous protein antigenic sites by comprehensive synthetic surface scanning: antibody and T-cell activity to several influenza hemagglutinin synthetic sites.

The determination in this laboratory of the complete antigenic structures of several proteins initially relied on a multi-approach complex chemical strategy and revealed that antigenic sites are surface locations which could be either 'continuous' or 'discontinuous' in architecture. More recently, we introduced a simplified comprehensive synthetic approach for the localization of the continuous antigenic sites of a protein. The approach depends on the synthesis of consecutive overlapping peptides, of uniform size and overlaps, and encompass the entire protein chain, from the beginning to the end. The latter approach is rather costly and labor-intensive, especially when applied to large protein molecules. All these studies showed, however, that protein antigenic sites occupy surface areas on a protein molecule. In order to render the determination of protein antigenic sites more feasible within a reasonable period of time, we considered that only the protein surface needs to be examined. Thus, for a protein of known three-dimensional structure, the protein surface can be readily screened for the continuous antigenic sites by the systematic synthesis and examination of immunochemical activity of all exposed segments of the protein. We have applied this approach here to influenza A virus hemagglutinin. Twelve peptides (11 reported for the first time here, and one reported previously), representing continuous surface segments of the molecule, have so far been synthesized, purified, characterized and their immunochemical activity studied. The peptides were found to bind anti-viral antibodies raised in outbred mice and antibodies in human sera from individuals that had suffered a recent influenza A infection. In one mouse strain (Balb/c; H-2d) so far examined, several of the peptides stimulated an in vitro proliferative response of T cells from virus (X-31)- primed mice. Finally, antisera to the peptides were raised in mice and, as expected, were found to bind to intact virus. In most cases, anti-peptide antibodies, did not bind disrupted virus. These studies indicate that protein 'continuous' antigenic sites can be localized by systematic synthetic scanning of the surface. It is emphasized that the approach is useful only for the localization of 'continuous' sites. The results also reveal that the antigenic structure of influenza virus hemagglutinin is more complex than has hitherto been suspected.

Structurally inherent antigenic sites. Localization of the antigenic sites of the alpha-chain of human haemoglobin in three host species by a comprehensive synthetic approach.

The antigenic structure of the alpha-chain of human haemoglobin was studied by a synthetic approach consisting of the synthesis of a series of consecutive overlapping peptides that together systematically represent the entire primary structure of the protein. This approach enabled the identification of a full profile of immunochemically active alpha-chain peptides and the localization of its major 'continuous' antigenic sites. Antibodies to haemoglobin raised in each of three different species (goat, rabbit and mouse) recognize similar sites on the alpha-chain. Further, the molecular locations of these sites coincide with alpha-chain regions extrapolated from antigenic sites of the conformationally similar myoglobin molecule. These findings support our earlier proposed concept of 'structurally inherent antigenic sites', namely that antigenicity is conferred on certain surface regions of proteins by virtue of their three-dimensional locations. Thus the antigenic sites of conformationally related proteins are likely to have similar molecular locations.

A novel and comprehensive synthetic approach for the elucidation of protein antigenic structures. Determination of the full antigenic profile of the alpha-chain of human haemoglobin.

A comprehensive synthetic approach for the determination of continuous antigenic sites of proteins is presented. This approach consists of the synthesis of a series of consecutive overlapping peptides that, together, systematically represent the entire primary structure of the protein under study. Its application to the alpha-chain of human haemoglobin afforded, for the first time, a full profile of immunochemically active alpha-chain peptides and enabled the localization of all the major continuous antigenic sites of this haemoglobin subunit.