Solid-phase Peptide Synthesis Method Research Articles

Preparation of a Tat-Related Transporter Peptide for Carrying the Adenovirus Vector into Cells

We synthesized a Tat-related peptide acetyl-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln-Gly-Cys amide, Ac-Tat(48-60)-Gly-Cys-NH(2), having high intracellular permeability, and conjugated this peptide to adenovirus vector to enhance gene transfer efficiency of adenovirus vector into cells. The peptide was prepared by the solid-phase peptide synthesis method and a bifunctional crosslinker 6-maleimidohexanoic acid N-hydroxysuccinimide ester was used to conjugate the peptide to adenovirus vector containing luciferase gene. The novel conjugate of adenovirus vector and Ac-Tat(48-60)-Gly-Cys-NH(2) peptide exhibited excellent gene transfer efficacy in B16BL6 cells.

Oxytocin analogues with amide groups substituted by tetrazole groups in position 4, 5 or 9.

Eleven oxytocin analogues substituted in position 4, 5 or 9 by tetrazole analogues of amino acids were prepared using solid-phase peptide synthesis method and tested for rat uterotonic in vitro and pressor activities, as well as for their affinity to human oxytocin receptor. The tetrazolic group has been used as a bioisosteric substitution of carboxylic, ester or amide groups in structure-activity relationship studies of biologically active compounds. Replacement of the amide groups of Gln(4) and Asn(5) in oxytocin by tetrazole analogues of aspartic, glutamic and alpha-aminoadipic acids containing the tetrazole moiety in the side chains leads to analogues with decreased biological activities. Oxytocin analogues in which the glycine amide residue in position 9 was substituted by tetrazole analogues of glycine had diminished activities as well. The analysis of differences in rat uterotonic activity and in the affinity to human oxytocin receptors of analogues containing either an acidic 5-substituted tetrazolic group or a neutral 1,5- or 2,5-tetrazole nucleus makes it possible to draw some new conclusions concerning the role of the amide group of amino acids in positions 4, 5 and 9 of oxytocin for its activity. The data suggest that the interaction of the side chain of Gln(4) with the oxytocin receptor is influenced mainly by electronic effects and the hydrogen bonding capacity of the amide group. Steric effects of the side chain are minor. Substitution of Asn(5) by its tetrazole derivative gave an analogue of very low activity. The result suggests that in the interaction between the amide group of Asn(5) and the binding sites of oxytocic receptor hydrogen bonds are of less importance than the spatial requirements for this group.

New Copper(II)/Cyclic Tetrapeptide System That Easily Oxidizes to Copper(III) under Atmospheric Oxygen

A 13-membered ring cyclic tetrapeptide was synthesized by the solid-phase peptide synthesis method, and its copper(II) coordination properties were analyzed by optical spectroscopy, mass spectrometry, and electrochemistry. All collected data strongly support the presence, at alkaline pH, of a stable peptide/copper(III) complex that is formed in solution by atmospheric dioxygen oxidation. On the basis of previous studies on cyclic peptide/copper systems, we suggest that the copper(III) ion is at the center of the ligand's cavity being coordinated to four deprotonated amide nitrogen atoms. This donor set would greatly lower the redox potential for the CuIII/CuII couple, thus allowing easy oxidation of the coordinated copper(II) by atmospheric oxygen.

Lytic Antimicrobial Activity of Hen Egg White Lysozyme Immobilized to Polystyrene Beads

Lysozyme [EC 3.2.1.17] was covalently attached to polystyrene resin beads by the sole histidine residue (His-15) through peptide spacers of various lengths. The spacers were amino acid chains composed of 6-aminocaproic acid synthesized with the solid phase peptide synthesis method. Immobilized lysozyme with a spacer length of three 6-aminocaproic acid units (2736 U/g resin with a protein load of 2.21 mg/g resin) displayed the greatest degree of hydrolytic activity against lyophilized Micrococcus lysodeikticus cell wall preparations. Enzymatic activity of immobilized lysozyme was 14.2% of that of the free enzyme. Preparations with longer spacers yielded higher total activity yet the retained activity was constant at about 14% level. A control that consisted of randomly coupled lysozyme to polystyrene beads without an amino acid spacer gave an enzyme activity of 158 U/g with a protein load of 1.24 mg/g resin which equated to 1.4% retained activity. Properties of the immobilized lysozyme system were studied, including stability and activity against soluble compared with insoluble substrates. A kinetics study of the immobilized lysozyme using Eadie-Hofstee plot parameters suggested significant external diffusion effects indicative of deviation from classic Michaelis-Menten kinetic behavior.

Variegin, a Novel Fast and Tight Binding Thrombin Inhibitor from the Tropical Bont Tick

Tick saliva contains potent antihemostatic molecules that help ticks obtain their enormous blood meal during prolonged feeding. We isolated thrombin inhibitors present in the salivary gland extract from partially fed female Amblyomma variegatum, the tropical bont tick, and characterized the most potent, variegin, one of the smallest (32 residues) thrombin inhibitors found in nature. Full-length variegin and two truncated variants were chemically synthesized. Despite its small size and flexible structure, variegin binds thrombin with strong affinity (K(i) approximately 10.4 pM) and high specificity. Results using the truncated variants indicated that the seven residues at the N terminus affected the binding kinetics; when removed, the binding characteristics changed from fast to slow. Further, the thrombin active site binding moiety of variegin is in the region of residues 8-14, and the exosite-I binding moiety is within residues 15-32. Our results show that variegin is structurally and functionally similar to the rationally designed thrombin inhibitor, hirulog. However, compared with hirulog, variegin is a more potent inhibitor, and its inhibitory activity is largely retained after cleavage by thrombin.

Industrial Applications of Solid-Phase Peptide Synthesis – A Status Report

Despite the fact that the structure of peptides has been known for more than a century, it was not until du Vigneaud published the synthesis of oxytocin 50 years later that the field was truly launched and the use of peptides as pharmaceuticals began. Since then, technical progress in the field has been astonishing, and the synthesis of peptides of virtually any size and complexity is now possible, with scale-up to the level of metric tonnes a reality. Perhaps the most important development was Merrifield’s publication of the solid-phase peptide synthesis (SPPS) method, which completely revolutionized the field, both from the perspective of accelerating research and discovery, and also because of its now widespread use for the manufacture of peptides for use as active pharmaceutical ingredients (APIs). The application of the SPPS method to the manufacture of peptide APIs will be reviewed, including both a historical overview and a summary of the current status. Some of the remaining challenges will also be discussed.

The Preparation of Solid-Supported Peptide Boronic Acids Derived from 4-Borono-L-phenylalanine and their Affinity for Alizarin

A library of solid-supported pentapeptide diboronic acids, a ‘lysine series’ and an ‘arginine series’, has been efficiently prepared using N-Fmoc-4-pinacolatoborono-l-phenylalanine and standard solid phase peptide synthesis methods. A technique for measuring the affinity of the chromophoric diol, alizarin, to the solid-supported peptide boronic acids has been developed. Considerable variation in alizarin binding strengths, both within and between arginine and lysine series was observed, with association constants in the range 200–1100 M–1 being recorded. The selective binding characteristics of these boronic acid–peptide hybrids suggest their potential use in carbohydrate sensors and cell-specific diagnostics and therapeutics.

Application of Cell Penetrating Peptide in Magnetic Resonance Imaging of Bone Marrow Mesenchymal Stem Cells

Tracking the distribution and differentiation of stem cells by high-resolution imaging techniques would have significant clinical and research implications. In this study, a model cell-penetrating peptide was used to carry gadolinium particles for magnetic resonance imaging (MRI) of mesenchymal stem cells (MSCs). MSCs were isolated from rat bone marrow and identified by osteogenic differentiation in vitro. The cell-penetrating peptide labeled with fluorescein-5-isothiocyanate (FITC) and gadolinium was synthesized by a solid-phase peptide synthesis method. Fluorescein imaging analysis confirmed that this new peptide could internalize into the cytoplasm and nucleus at room temperature, 4 degrees and 37 degrees . Gadolinium were efficiently internalized into mesenchymal stem cells by the peptide in a time or concentration-dependent manner, resulting in intercellular shortening of longitudinal relaxation enhancements, which were obviously detected by 1.5 Tesla Magnetic Resonance Imaging. Cytotoxicity assay and flow cytometric analysis showed that the intercellular contrast medium incorporation did not affect cell viability at the tested concentrations. The in vitro experiment results suggested that the new constructed peptides could be a vector for tracking MSCs.

A Facile Synthesis and Physical Properties of Nano-Sized Dendritic α,ε-Poly(L-lysine)s for the Delivery of Nucleic Acids

A series of nano-sized dendritic alpha,epsilon-poly(L-lysine)s (DPL) were synthesized by the solid-phase peptide synthesis method, using a core epsilon-peptide structure consisting of eight lysine residues. Surface amines of dendritic alpha,epsilon-poly(L-lysine) were characterized by comparing the retention times of a reverse phase HPLC with the electrophoretic mobilities of capillary zone electrophoresis (CZE) and non-denatured polyacrylamide gel electrophoresis (PAGE). The elution times of alpha,epsilon-poly(Llysine) in HPLC were well correlated with the electrophoretic mobilities of CZE and PAGE. The separation was dependent on size, shape of molecule and the number of surface amine. The alpha,epsilon-poly(L-lysine) formed a complex with nucleic acids at various charge ratios and the degree of complex formation was size- and structure-dependent. Atomic force microscopy of the complex visualized the size and morphology of alpha,epsilon-poly(L-lysine)/DNA complex as a nano-sized spherical shape. The small size in complex formation provides a promise for in vivo therapeutic application of dendritic alpha,epsilon-poly(L-lysine)s or their derivatives in the delivery of gene or oligonucleotide.

A Facile Synthesis and Physical Properties of Nano-Sized Dendritic <I>α</I>,<I>ε</I>-Poly(L-lysine)s for the Delivery of Nucleic Acids

A series of nano-sized dendritic alpha,epsilon-poly(L-lysine)s (DPL) were synthesized by the solid-phase peptide synthesis method, using a core epsilon-peptide structure consisting of eight lysine residues. Surface amines of dendritic alpha,epsilon-poly(L-lysine) were characterized by comparing the retention times of a reverse phase HPLC with the electrophoretic mobilities of capillary zone electrophoresis (CZE) and non-denatured polyacrylamide gel electrophoresis (PAGE). The elution times of alpha,epsilon-poly(Llysine) in HPLC were well correlated with the electrophoretic mobilities of CZE and PAGE. The separation was dependent on size, shape of molecule and the number of surface amine. The alpha,epsilon-poly(L-lysine) formed a complex with nucleic acids at various charge ratios and the degree of complex formation was size- and structure-dependent. Atomic force microscopy of the complex visualized the size and morphology of alpha,epsilon-poly(L-lysine)/DNA complex as a nano-sized spherical shape. The small size in complex formation provides a promise for in vivo therapeutic application of dendritic alpha,epsilon-poly(L-lysine)s or their derivatives in the delivery of gene or oligonucleotide.

Combinatorial Solid‐Phase Peptide Synthesis and Bioassays

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Paramagnetic particles carried by cell-penetrating peptide tracking of bone marrow mesenchymal stem cells, a research in vitro

The ability to track the distribution and differentiation of stem cells by high-resolution imaging techniques would have significant clinical and research implications. In this study, a model cell-penetrating peptide was used to carry gadolinium particles for magnetic resonance imaging of the mesenchymal stem cells. The mesenchymal stem cells were isolated from rat bone marrow by Percoll and identified by osteogenic differentiation in vitro. The cell-penetrating peptides labeled with fluorescein-5-isothiocyanate and gadolinium were synthesized by a solid-phase peptide synthesis method and the relaxivity of cell-penetrating peptide-gadolinium paramagnetic conjugate on 400 MHz nuclear magnetic resonance was 5.7311 ± 0.0122 mmol −1 s −1, higher than that of diethylenetriamine pentaacetic acid gadolinium ( p < 0.05). Fluorescein imaging confirmed that this new peptide could internalize into the cytoplasm and nucleus. Gadolinium was efficiently internalized into mesenchymal stem cells by the peptide in a time- or concentration-dependent fashion, resulting in intercellular T1 relaxation enhancement, which was obviously detected by 1.5 T magnetic resonance imaging. Cytotoxicity assay and flow cytometric analysis showed the intercellular contrast medium incorporation did not affect cell viability and membrane potential gradient. The research in vitro suggests that the newly constructed peptides could be a vector for tracking mesenchymal stem cells.

Total chemical synthesis of the B1 domain of protein L from Peptostreptococcus magnus

Reported here is a native chemical ligation strategy for the total chemical synthesis of the B1 domain of protein L. A synthetic construct of this 76 amino acid protein domain was prepared by the chemoselective ligation of two unprotected polypeptide fragments, one containing an N-terminal cysteine residue and one containing a C-terminal thioester moiety. The polypeptide fragments utilized in the ligation reaction were readily prepared by stepwise solid phase peptide synthesis (SPPS) methods for Boc-chemistry. The milligram quantities of protein required for conventional biophysical studies were readily accessible using the synthetic protocol described here. The folding properties of the synthetic protein L construct were also determined and found to be very similar to those of a similar wild-type protein L constructs prepared by recombinant-DNA methods. This work facilitates future unnatural amino acid mutagenesis experiments on this model protein system to further dissect the molecular basis of its folding and stability.

Proteomics analysis of a novel compound: Cyclic RGD in breast carcinoma cell line MCF-7

In studies of cell adhesion, migration, growth, differentiation, and apoptosis, synthetic peptides containing the RGD (Arg-Gly-Asp) motif have been extensively used as the inhibitors of integrin-ligand interactions. The RGD motif is an integrin-recognition motif found in many ligands, so that the RGD-containing peptides can be used to probe integrin functions in various biological systems. A linear RGD is a tripeptide consisting of a flexible structure that makes the motif bind to its receptor with inefficient chelating affinity. Therefore, we designed a cyclic-RGD peptide (Tpa-RGDWPC, cRGD) with rigid skeleton to closely bind with its receptor. The cRGD was obtained by solid-phase peptide synthesis method using Rink amide resin. We showed that the cRGD exerts more potency than linear RGD on inhibiting cell growth of MCF-7 breast carcinoma cells. This stimulated us to question how cRGD inhibits cell growth of MCF-7 cells. Moreover, understanding what molecular mechanism underlies the effect that RGD motif exerts on MCF-7 cells is also of considerable importance. We used proteomics and bioinformatics to survey the global changes in proteins after cRGD treatment in MCF-7 cells. The classification of these proteins is shown according to the different biological processes in which they are involved. Most of the proteins that appear to be strongly influenced by cRGD treatment are involved in metabolism, cell growth, responsive to external stimulus, cell communication, reproduction and cell death. This is the first report which monitors the protein expression profile of MCF-7 cells in response to treatment with RGD-containing peptides in a time-course analysis. The clustering data indicated temporal patterns of altered protein expression that can be categorized into early, intermediate and late response proteins. These patterns of protein expression may be important for predicting its response to cRGD. In summary, these results provide a molecular explanation for the properties of cRGD in breast cancer cells and present a valuable in-depth description of their possible impact on breast cancer therapy.

A novel solid-phase linker strategy for the side-chain anchoring of arginine: an expeditious route to arginine 7-amido-4-methylcoumarins

A novel linker strategy for the efficient side-chain anchoring of arginine is described. The utility of this approach was demonstrated by the facile synthesis of arginine-specific fluorogenic peptide substrates by standard Fmoc solid phase peptide synthesis methods.

Synthesis of 3β [L-Lysinamide-carbamoyl] Cholesterol Derivatives by Solid-Phase Method and Characteristics of Complexes with Antisense Oligodeoxynucleotides

In this report, we describe the synthesis of mono- and di-valent cationic [L-Lysinamide-carbamoyl] cholesterol (K-Chol) derivatives by solid-phase peptide synthesis method and the characteristics of K-Chol/antisense oligodeoxynucleotide (ODN) complexes. K-Chol was able to interact with antisense ODNs electrostatically and constructed nanometer-sized complexes of 50-100 nm in diameter. The formation of K-Chol/antisense ODN complexes was demonstrated by non-denaturing polyacrylamide gel electrophoresis assay and atomic force microscopy. The cell-associated radioactivity was measured to monitor the cellular uptake of the complexes containing radioactive antisense ODNs using HL 60 cells.

Combinatorial Solid Phase Peptide Synthesis and Bioassays

Solid phase peptide synthesis method, which was introduced by Merrifield in 1963, has spawned the concept of combinatorial chemistry. In this review, we summarize the present technologies of solid phase peptide synthesis (SPPS) that are related to combinatorial chemistry. The conventional methods of peptide library synthesis on polymer support are parallel synthesis, split and mix synthesis and reagent mixture synthesis. Combining surface chemistry with the recent technology of microelectronic semiconductor fabrication system, the peptide microarray synthesis methods on a planar solid support are developed, which leads to spatially addressable peptide library. There are two kinds of peptide microarray synthesis methodologies: pre-synthesized peptide immobilization onto a glass or membrane substrate and in situ peptide synthesis by a photolithography or the SPOT method. This review also discusses the application of peptide libraries for high-throughput bioassays, for example, peptide ligand screening for antibody or cell signaling, enzyme substrate and inhibitor screening as well as other applications.

Sugar Amino Acids and Related Molecules: Some Recent Developments

AbstractFor Abstract see ChemInform Abstract in Full Text.

Synthesis and bioactivities of two multiple antigen peptides as potential vaccine against schistosoma

Based on the two antigenic peptides, 26–43 (P 26) and 116–131 (P 116), derived from 28 kDa glutathione S-transferase of Schistosoma mansoni (Sm28GST), two multiple antigenic peptides (MAPs), (P 26) 4-MAP and (P 116) 4-MAP with the same oligomeric lysine core, were synthesized by stepwise solid-phase peptide synthesis method. The antigenicities and protective effects of these two MAPs were examined on experimental animals. As shown in the dot-ELISA result, the synthetic MAPs could be recognized and bound by immunoglobins in both patient’s and infected-rabbit’s sera. After Kunming mice were immunized with (P 26) 4-MAP, the worm burden reduction rate and the liver egg reduction rate were 59.9% and 61.1%. In (P 26) 4-MAP or (P 116) 4-MAP immunized BALB/c mice, the worm burden reduction rates were 37.5% and 62.5%, respectively, and the liver egg reduction rates were 35.1% and 54.0%, respectively.

Sugar amino acids and related molecules: Some recent developments

To meet the growing demands for the development of new molecular entities for discovering new drugs and materials, organic chemists have started working on many new concepts that can help to assimilate knowledge-based structural diversities more efficiently than ever before. Emulating the basic principles followed by Nature to build its vast repertoire of biomolecules, organic chemists are developing many novel multifunctional building blocks and using them to create ‘nature-like’ and yet unnatural organic molecules. Sugar amino acids constitute an important class of such polyfunctional scaffolds where the carboxyl, amino and hydroxyl termini provide an excellent opportunity to organic chemists to create structural diversities akin to Nature’s molecular arsenal. In recent years, sugar amino acids have been used extensively in the area of peptidomimetic studies. Advances made in the area of combinatorial chemistry can provide the necessary technological support for rapid compilations of sugar amino acidbased libraries exploiting the diversities of their carbohydrate frameworks and well-developed solid-phase peptide synthesis methods. This perspective article chronicles some of the recent applications of various sugar amino acids, furan amino acids, pyrrole amino acids etc. and many other related building blocks in wide-ranging peptidomimetic studies