Stepwise Solid-phase Synthesis Research Articles

Total stepwise solid-phase synthesis of peptide-oligonucleotide conjugates using side-chain Boc/tBu protecting groups.

A practical strategy for the total stepwise solid-phase synthesis of peptide-oligonucleotide conjugates was developed. In this strategy, the Boc/tBu protecting groups are utilized for the side chains of Trp, His, Arg, Asp, and Glu, and is deprotected in borate buffer at 90 °C to avoid depurination of the oligonucleotide caused by strong acid treatment. The advantage of this strategy is that the abovementioned amino acids are readily available in the market and the side reaction of deguanidination of the Arg residue can be avoided. This side-chain Boc/tBu protection strategy will expand the applicability of total stepwise synthesis in the preparation of peptide-oligonucleotide conjugates.

Covalent Attachment and Detachment by Reactive DESI of Sequence-Coded Polymer Taggants.

The use of sequence-defined polymers is an interesting emerging solution for materials identification and traceability. Indeed, a very large amount of identification sequences can be created using a limited alphabet of coded monomers. However, in all reported studies, sequence-defined taggants are usually included in a host material by noncovalent adsorption or entrapment, which may lead to leakage, aggregation, or degradation. To avoid these problems, sequence-defined polymers are covalently attached in the present work to the mesh of model materials, namely acrylamide hydrogels. To do so, sequence-coded polyurethanes containing a disulfide linker and a terminal methacrylamide moiety are synthesized by stepwise solid-phase synthesis. These methacrylamide macromonomers are afterward copolymerized with acrylamide and bisacrylamide in order to achieve cross-linked hydrogels containing covalently-bound polyurethane taggants. It is shown herein that these taggants can be selectively detached from the hydrogel mesh by reactive desorption electrospray ionization. Using dithiothreitol the disulfide linker that links the taggant to the gel can be selectively cleaved. Ultimately, the released taggants can be decoded by tandem mass spectrometry.

Synthesis of MUC1 Peptide Backbone Bearing Sialyl‐Tn Antigen for Structural and Functional Studies With Endogenous Lectins

ObjectiveTo synthesize the tumor associated analogs of MUC1 for use in structural and functional studies between sialyl‐Tn (sTn) antigen with macrophage galactose‐specific lectin (MGL).AbstractMucin 1 (MUC1) is a heavily glycosylated transmembrane protein, altered in both expression and glycosylation pattern in human carcinomas of the epithelium. The presence of incomplete or truncated glycan structures, often capped by sialic acid, commonly known as tumor‐associated carbohydrate antigens (TACAs), on the cell surface is a well‐known cancer biomarker. Accumulating evidence suggests that TACAs expression is associated with tumor escape from immune defenses through interaction with endogenous carbohydrate binding proteins (lectins). These interactions often lead to creation of a protumor microenvironment, favoring tumor initiation, progression, metastasis, and immune evasion. Macrophage galactose binding lectin (MGL) is a C‐type lectin receptor found on antigen‐presenting cells (APCs) which facilitates the uptake of carbohydrate antigens for antigen presentation, modulating the immune response homeostasis, autoimmunity, and cancer. Considering the crucial role of tumor‐associated forms of MUC1 with MGL in tumor immunology, a thorough understanding of this interaction is essential for it to be exploited for cancer vaccine strategies. The specific aim of this research is to synthesize structurally well‐defined chemical probes, mono and multiple glycosylated MUC1 glycopeptide models carrying the Tn or sTn, that allow for the control of the complexity of the chemical space of the multivalent ligands. For this purpose, a concise scheme was developed for the large synthesis of the sTn antigen building block. Thiophenyl glycoside donors, in the presence TfOH/NIS or TMSOTf/NIS as promoter system, were used for the galactosylation and sialylation steps of the amino acid building block synthesis, respectively. We explored the effect of activator, temperature, solvent, and excess equivalent of sialic acid thioglycoside donor on sialylation reaction. The most favorable reaction conditions improved the desired α‐stereoselectivity, decreased the competing 2,3‐elimination reaction on the sialyl donor during activation, leading to overall increase in the reaction yield. This building block was used in the stepwise solid‐phase peptide synthesis of MUC1 glycopeptide models. The secondary structure of synthesized peptides was determined by circular dichroism (CD) spectroscopy.

Chemistry of Peptide-Oligonucleotide Conjugates: A Review.

Peptide-oligonucleotide conjugates (POCs) represent one of the increasingly successful albeit costly approaches to increasing the cellular uptake, tissue delivery, bioavailability, and, thus, overall efficiency of therapeutic nucleic acids, such as, antisense oligonucleotides and small interfering RNAs. This review puts the subject of chemical synthesis of POCs into the wider context of therapeutic oligonucleotides and the problem of nucleic acid drug delivery, cell-penetrating peptide structural types, the mechanisms of their intracellular transport, and the ways of application, which include the formation of non-covalent complexes with oligonucleotides (peptide additives) or covalent conjugation. The main strategies for the synthesis of POCs are viewed in detail, which are conceptually divided into (a) the stepwise solid-phase synthesis approach and (b) post-synthetic conjugation either in solution or on the solid phase, especially by means of various click chemistries. The relative advantages and disadvantages of both strategies are discussed and compared.

Optimized Stepwise Synthesis of the API Liraglutide Using BAL Resin and Pseudoprolines.

The number of peptide-based active pharmaceutical ingredients (APIs) has increased enormously in recent years. Furthermore, the emerging new peptide drug candidates are more complex and larger. For the industrial solid-phase synthesis of C-carboxylic acid peptides, the two main resins available, Wang and chlorotrityl chloride (CTC), have a number of drawbacks. In this context, resins that form an amide bond with the first amino acid are more robust than Wang and CTC resins. Here, we address the use of the backbone (BAL) resin for the synthesis of the peptide liraglutide. The BAL resin, in conjunction with the use of pseudoprolines to avoid aggregation, allows the stepwise solid-phase synthesis of this API in excellent purity and yield.

New Generation of Clickable Nucleic Acids: Synthesis and Active Hybridization with DNA.

Due to the ability to generate oligomers of precise sequence, sequential and stepwise solid-phase synthesis has been the dominant method of producing DNA and other oligonucleotide analogues. The requirement for a solid support, however, and the physical restrictions of limited surface area thereon significantly diminish the efficiency and scalability of these syntheses, thus, negatively affecting the practical applications of synthetic polynucleotides and other similarly created molecules. By employing the robust photoinitiated thiol-ene click reaction, we developed a new generation of clickable nucleic acids (CNAs) with a polythioether backbone containing repeat units of six atoms, matching the spacing of the phosphodiester backbone of natural DNA. A simple, inexpensive, and scalable route was utilized to produce CNA monomers in gram-scale, which indicates the potential to dramatically lower the cost of these DNA mimics and thereby expand the scope of these materials. The efficiency of this approach was demonstrated by the completion of CNA polymerization in 30 seconds, as characterized by size-exclusive chromatography (SEC) and infrared (IR) spectroscopy. CNA/DNA hybridization was demonstrated by gel electrophoresis and used in CdS nanoparticle assembly.

One-Pot Peptide Ligation-Oxidative Cyclization Protocol for the Preparation of Short-/Medium-Size Disulfide Cyclopeptides.

Native chemical ligation (NCL) employing the N-methylbenzimidazolinone (MeNbz) linker readily provided the linear precursor of a 16-mer peptide that is difficult to obtain by stepwise solid-phase peptide synthesis. NCL and the workup conditions were improved toward a protocol that allows for quantitative removal of the 4-hydroxymercaptophenol additive and subsequent formation of the disulfide bridge in the NCL cocktail by oxidation in air, tolerated by the presence of tris(hydroxypropyl)phosphine.

Mambalgins, snake peptides against inflammatory and neuropathic pain through inhibition of ASIC channels

Mambalgins are 57-amino acid peptides isolated from mamba venom. They produce potent analgesic effects in mice against inflammatory pain upon central intrathecal (i.t.), and peripheral local (i.pl.) injections, through inhibition of different ASICs subtypes and involvement of opioid-independent pathways. They produce fewer side effects than morphine and no apparent toxicity. We now show that mambalgins also have an opioid-independent effect on both thermal and mechanical pain upon systemic intravenous (i.v.) administration and are effective against neuropathic pain by i.v., i.t. and i. pl. injections. By combining the use of knockdown and knockout animals, we show the critical involvement of peripheral ASIC1b-containing channels in the i.v. effects of mambalgins against inflammatory pain. The potent analgesic effect on neuropathic pain involves two different mechanisms depending on the route of administration, a naloxone-insensitive and ASIC1a-independent effect associated with i.v. injection, and an ASIC1a-dependent and partially naloxone-sensitive effect associated with i.t. injection. We have done in collaboration with the CEA iBiTecS Institute in Gif-sur-Yvette, the full stepwise solid-phase peptide synthesis of mambalgin-1, solved its 3D crystal structure, mapped the pharmacophore, and identified the binding site and the inhibitory mechanism on ASIC1a channels. These findings identify new roles for ASICs in pain pathways, and mambalgins as new potential analgesics against inflammatory and chronic neuropathic pain.

Mambalgin-1 Pain-relieving Peptide, Stepwise Solid-phase Synthesis, Crystal Structure, and Functional Domain for Acid-sensing Ion Channel 1a Inhibition

Mambalgins are peptides isolated from mamba venom that specifically inhibit a set of acid-sensing ion channels (ASICs) to relieve pain. We show here the first full stepwise solid phase peptide synthesis of mambalgin-1 and confirm the biological activity of the synthetic toxin both in vitro and in vivo. We also report the determination of its three-dimensional crystal structure showing differences with previously described NMR structures. Finally, the functional domain by which the toxin inhibits ASIC1a channels was identified in its loop II and more precisely in the face containing Phe-27, Leu-32, and Leu-34 residues. Moreover, proximity between Leu-32 in mambalgin-1 and Phe-350 in rASIC1a was proposed from double mutant cycle analysis. These data provide information on the structure and on the pharmacophore for ASIC channel inhibition by mambalgins that could have therapeutic value against pain and probably other neurological disorders.

Novel bone scaffold containing biomimetic peptide for bone tissue engineering

Event Abstract Back to Event Novel bone scaffold containing biomimetic peptide for bone tissue engineering Samaneh Hosseini1, Mohamadreza Baghaban Eslaminejad1 and Shahab Faghihi2 1 Royan Institute for Stem Cell Biology and Technology, ACECR, Department of Stem Cells and Developmental Biology, Cell Science Research Center, Iran 2 National Institute of Genetic Engineering and Biotechnology, Tissue Engineering and Biomaterials Division, Iran The regeneration of diseased or fractured bone represents a premier clinical need, as numerous patients suffer from deficient healing of bone in variety of medical conditions like traumatic fracture, congenital malformation, and surgically bone defects. Tissue engineering emerges as a promising strategy to overcome limitations of traditional bone grafts such as donor site morbidity and the absence of osteogenic properties [1]. Biomaterial design with biomimetic peptide which have biomineralization ability holds great potential for successful bone regeneration. Since the hydroxyapatite (HA) is the major inorganic component of bone, HA-binding ‎peptide could promote formation of mineral phase of bone and osteogenesis [2][3]. Our previous study showed that a natural motif sequence of osteocalcin (OSN) has ability to nucleate apatite nanocrystals[4]. The objective of this study is to develop a collagen-chitosan nanoparticle containing biomineralizing peptide scaffold for directing the osteogenic differentiation of adipose derived stem cells (AD-MSCs). Toward this goal, osteocalcin mimetic peptide (OSN) was constructed by stepwise solid-phase synthesis manually. The peptide was purified by reverse-phase HPLC and was checked for ‎homogeneity by analytical RP-HPLC which indicated a purity> 95%. Molecular weights was ‎confirmed by MALDI-TOF mass ‎spectra.‎ Peptide-chitosan nanoparticles were prepared by physical self-assembly method. Surface charge and mean particle ‎size of peptide and peptide-nanoparticles were determined by ‎‎nanozetasizer. OSN-chitosan nanoparticles were networked with collagen to provide multifunctional and structurally stable biomatrix, following were analyzed using scanning electron microscopy (SEM). Adipose derived stem cells were isolated from the fat tissue of 6 weeks Wistar rats and were then characterized for osteoblast, adipocyte and chondrocytes differentiations. ‎‎‎‎The ‎cellular response of AD-MSCs on proliferation and viability is ‎examined using MTT assay. Mineralization of extracellular ‎matrix and accumulation of calcium was also assessed using Alizarin red-S staining and calcium ‎assay. Evaluation of constructed scaffold revealed the optimum structural properties. MTT results confirmed that not only is there no evidence of cytotoxicity related to OSN but also the cells which are cultured on OSN containing sample had ‎significant increase in proliferation rate in comparison with control (Fig. 1). Calcium content analysis indicated that there was a 2-fold increase in the amount of calcium in the presence of OSN compared ‎to control. According to alizarin S staining, the number of mineralized nodules was increased in samples containing ‎OSN compared with control (Fig. 2). Altogether, it is believed that the construct containing OSN is able to induce bone formation by increasing mineralization. This ‎study has significant implication for tissue engineering and osteocalcin mimetic peptide may have great utility in future bone clinical applications as a bioactive inducer of biomineralization for bone regeneration.

Lipopeptide Nanoparticles: Development of Vaccines against Hookworm Parasite.

Necator americanus (hookworm) infects over half a billion people worldwide. Anthelminthic drugs are commonly used to treat the infection; however, vaccination is a more favorable strategy to combat this parasite. We designed new B-cell peptide epitopes based on the aspartic protease of N. americanus (Na-APR-1). The peptides were conjugated to self-adjuvanting lipid core peptide (LCP) systems via stepwise solid-phase peptide synthesis (SPPS) and copper catalyst azide-alkyne cycloaddition (CuAAC) reactions. The LCP vaccine candidates were able to self-assemble into nanoparticles, were administered to mice without the use of additional adjuvant, and generated antibodies that recognized the parent epitope. However, only one LCP derivative was able to produce a high titer of antibodies specific to Na-APR-1; circular dichroism analyses of this compound showed a β-sheet conformation for the incorporated epitope. This study provides important insight in epitope and delivery system design for the development of a vaccine against hookworm infections.

Increased Diversity of the HLA-B40 Ligandome by the Presentation of Peptides Phosphorylated at Their Main Anchor Residue

Human leukocyte antigen (HLA) class I molecules bind peptides derived from the intracellular degradation of endogenous proteins and present them to cytotoxic T lymphocytes, allowing the immune system to detect transformed or virally infected cells. It is known that HLA class I-associated peptides may harbor posttranslational modifications. In particular, phosphorylated ligands have raised much interest as potential targets for cancer immunotherapy. By combining affinity purification with high-resolution mass spectrometry, we identified more than 2000 unique ligands bound to HLA-B40. Sequence analysis revealed two major anchor motifs: aspartic or glutamic acid at peptide position 2 (P2) and methionine, phenylalanine, or aliphatic residues at the C terminus. The use of immobilized metal ion and TiO2 affinity chromatography allowed the characterization of 85 phosphorylated ligands. We further confirmed every sequence belonging to this subset by comparing its experimental MS2 spectrum with that obtained upon fragmentation of the corresponding synthetic peptide. Remarkably, three phospholigands lacked a canonical anchor residue at P2, containing phosphoserine instead. Binding assays showed that these peptides bound to HLA-B40 with high affinity. Together, our data demonstrate that the peptidome of a given HLA allotype can be broadened by the presentation of peptides with posttranslational modifications at major anchor positions. We suggest that ligands with phosphorylated residues at P2 might be optimal targets for T-cell-based cancer immunotherapy.

Fmoc-aza-β3-Lys-OAllyl and Fmoc-aza-β3-Asp-OAllyl for on-resin head-to-tail cyclization of aza-β3-peptides

A rapid and efficient Fmoc/t-Bu solid-phase peptide synthesis (SPPS) of cyclic aza-β3-peptide analogs by allyl strategy is described. Our synthetic approach includes the synthesis of two new aza-β3-amino acids, Fmoc-aza-β3-Lys-OAllyl and Fmoc-aza-β3-Asp-OAllyl, then the resin attachment of the first aza-β3-amino acid via its side chain, successful use of combination of three orthogonal removable protecting groups, stepwise solid-phase synthesis of linear peptide analog, and on-resin head-to-tail cyclization.

Solution-phase synthesis and evaluation of tetraproline chiral stationary phases

A protocol was developed for the solution-phase synthesis of multigram amounts of two 9-fluorenylmethoxycarbonyl (Fmoc)-protected tetraproline peptides. These tetraproline peptides were then attached to amino derivatized silica gel. The replacement of the Fmoc group with the trimethylacetyl group lead to two tetraproline chiral stationary phases (CSPs). A comparison of the chromatographic behavior of these two solution-phase-synthesized tetraproline CSPs with that prepared by stepwise solid-phase synthesis revealed that all three had similar chromatographic performance for resolving 53 model analytes. This suggests that the solution-phase synthesis of oligoprolines, which allows for the specific benefits of good batch reproducibility, selector homogeneity, and possibly low cost, is a feasible alternative to the solid-phase synthesis of oligoproline CSPs.

Anthelmintic PF1022A: stepwise solid-phase synthesis of a cyclodepsipeptide containing N-methyl amino acids

Cyclodepsipeptides of the enniation-, PF1022-, and verticilide-family represent a diverse class of highly interesting natural products with respect to their manifold biological activities. However, until now no stepwise solid-phase synthesis has been accomplished due to the difficult combination of N-methyl amino acids and hydroxycarboxylic acids. We report here the first stepwise solid-phase synthesis of the anthelmintic cyclooctadepsipeptide PF1022A based on an Fmoc/THP-ether protecting group strategy on Wang-resin. The standard conditions of our synthesis allow an unproblematic adaption to an automated peptide synthesizer.

Disulfide Bond Formation with Polymer-Supported Reagent

Oxidizing reagents for disulfide formation of peptides are prepared from poly(ethylene glycol)polyacrylamide (PEGA) via the stepwise solid-phase peptide synthesis (method 1) or the oligomerization of N-carboxy anhydride derivatives (method 2). The oxidation of peptides 1 and 2 was carried out with resins 1a-d and 2 to give the corresponding disulfides 1′ and 2′ in 90-100% yield.

A Synthetic S6 Segment Derived from KvAP Channel Self-assembles, Permeabilizes Lipid Vesicles, and Exhibits Ion Channel Activity in Bilayer Lipid Membrane

KvAP is a voltage-gated tetrameric K(+) channel with six transmembrane (S1-S6) segments in each monomer from the archaeon Aeropyrum pernix. The objective of the present investigation was to understand the plausible role of the S6 segment, which has been proposed to form the inner lining of the pore, in the membrane assembly and functional properties of KvAP channel. For this purpose, a 22-residue peptide, corresponding to the S6 transmembrane segment of KvAP (amino acids 218-239), and a scrambled peptide (S6-SCR) with rearrangement of only hydrophobic amino acids but without changing its composition were synthesized and characterized structurally and functionally. Although both peptides bound to the negatively charged phosphatidylcholine/phosphatidylglycerol model membrane with comparable affinity, significant differences were observed between these peptides in their localization, self-assembly, and aggregation properties onto this membrane. S6-SCR also exhibited reduced helical structures in SDS micelles and phosphatidylcholine/phosphatidylglycerol lipid vesicles as compared with the S6 peptide. Furthermore, the S6 peptide showed significant membrane-permeabilizing capability as evidenced by the release of calcein from the calcein-entrapped lipid vesicles, whereas S6-SCR showed much weaker efficacy. Interestingly, although the S6 peptide showed ion channel activity in the bilayer lipid membrane, despite having the same amino acid composition, S6-SCR was significantly inactive. The results demonstrated sequence-specific structural and functional properties of the S6 wild type peptide. The selected S6 segment is probably an important structural element that could play an important role in the membrane interaction, membrane assembly, and functional property of the KvAP channel.

Intrinsically Disordered Mollusk Shell Prismatic Protein That Modulates Calcium Carbonate Crystal Growth

The formation of calcite prism architecture in the prismatic layer of the mollusk shell involves the participation of a number of different proteins. One protein family, Asprich, has been identified as a participant in amorphous calcium carbonate stabilization and calcite architecture in the prismatic layer of the mollusk, Atrina rigida . However, the functional role(s) of this protein family are not fully understood due to the fact that insufficient quantities of these proteins are available for experimentation. To overcome this problem, we employed stepwise solid-phase synthesis to recreate one of the 10 members of the Asprich family, the 61 AA single chain protein, Asprich "3". We find that the Asprich "3" protein inhibits the formation of rhombohedral calcite crystals and induces the formation of round calcium carbonate deposits in vitro that contain calcite and amorphous calcium carbonate (ACC). This mineralization behavior does not occur under control conditions, and the formation of ACC and calcite is similar to that reported for the recombinant form of the Asprich "g" protein. Circular dichroism studies reveal that Asprich "3" is an intrinsically disordered protein, predominantly random coil (66%), with 20-30% β-strand content, a small percentage of β-turn, and little if any α-helical content. This protein is not extrinsically stabilized by Ca(II) ions but can be stabilized by 2,2,2-trifluoroethanol to form a structure consisting of turn-like and random coil characteristics. This finding suggests that Asprich "3" may require other extrinsic interactions (i.e., with mineral or ionic clusters or other macromolecules) to achieve folding. In conclusion, Asprich "3" possesses in vitro functional and structural qualities that are similar to other reported for other Asprich protein sequences.

Stepwise synthesis of oligonucleotide–peptide conjugates containing guanidinium and lipophilic groups in their 3′-termini

Two different series of oligonucleotide–peptide conjugates have been efficiently synthesized by stepwise solid-phase synthesis. First, oligonucleotides and oligonucleotide phosphorothioates containing polar groups at the 3′-termini, such as amine and guanidinium groups were prepared. ODNs conjugates carrying several lysine residues were obtained directly from Fmoc deprotection whereas ODN conjugates with guanidinium groups were obtained by post-synthetic guanidinylation. The second family contains different urea moieties that were achieved by standard protocols. All products were fully characterized by reversed phase HPLC and MALDI-TOF mass spectrometry yielding satisfactory results. Oligonucleotide–phosphorothioate conjugates were evaluated as potential antisense oligonucleotides in the inhibition of the luciferase gene.

Strategies and Limitations in Dendrimeric Immunogen Synthesis. The Influenza Virus M2e Epitope as a Case Study

Dendrimeric platforms such as multiple antigen peptides (MAPs) are regarded as one of the most efficacious approaches for antigenic presentation. Originally described as available by stepwise solid-phase peptide synthesis (SPPS), MAPs have also been prepared by chemical (thioether, oxime, hydrazone) ligation of appropriately functionalized tetra- or octavalent polylysine scaffolds with the peptide antigen to be multiply displayed. In this work, the advantages and limitations of two of the most frequent methods of MAP preparation, namely, chemoselective thioether ligation in solution, and all-solid-phase synthesis, have been tested in the case of a particularly troublesome epitope model, the ectodomain of protein M2 from influenza virus (M2e). The strong tendency of M2e to self-associate is a serious inconvenient for conjugation in solution, which as a result fails to produce the target MAPs with the specified number of M2e copies. In contrast, the fully stepwise SPPS approach is shown to be quite practical, especially when 6-aminohexanoic acid spacer units providing increased internal flexibility are inserted at each branching point.