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Fundamentals of Modern Peptide Synthesis
Published in Mesut Karahan, Synthetic Peptide Vaccine Models, 2021
Protein synthesis is important to study the structure of natural proteins, to investigate how protein structure and function are formed by the amino acid sequence and to understand the synthesis mechanism. However, it is not possible to form this structure by simply mixing the amino acids together. Protective groups are often necessary to avoid undesirable reactions. Chemical peptide synthesis generally starts from the carboxyl groups (C-terminus) of the peptide and proceeds towards the amino groups (N-terminus). This is the opposite direction of protein biosynthesis. The resulting bond is the amide (peptide) bond. It is necessary to use protective groups to control the C-N coupling reaction. Certain peptide bonds can be formed by protecting the amine group (N-terminus) of one amino acid and the carboxyl group (C-terminus) of the another. The protection of side chain functional groups is necessary to prevent undesirable reactions and to form univocally the goal peptide bond. The peptide bonds are also called eupeptide bonds to distinguish them from isopeptide bonds formed by participating side chain functional groups as is the case of glutathione for example. Peptide synthesis is a multistage process comprising a number of chemical processes. To provide an overall high yield of the goal peptide it is drastically important to have yields close to quantitative at all stages of this complex process. It is also extremely important to avoid even a low racemization at all stages otherwise the obtained peptide will lack the desirable physiological activity (Isidro-Llobet, Álvarez, and Albericio 2009).
Synthesis of Bioactive Peptides for Pharmaceutical Applications
Published in Peter Grunwald, Pharmaceutical Biocatalysis, 2019
Jaison Jeevanandam, Ashish Kumar Solanki, Shailza Sharma, Prabir Kumar Kulabhusan, Sapna Pahil, Michael K. Danquah
The common methods of peptide synthesis discussed in the previous sections are effective for enhanced peptide production. However, there exist challenges in the fabrication of certain sequences of peptides, mainly because of lesser rate of accomplishment with peptide complexity;improved chances of aggregation either inter- and/or intramolecular;inclined probability for the development of secondary structure and protecting groups that are hindered by steric arrangements which generate sequence for early termination. Thus, there are few novel or alternative techniques that have been developed in recent times as a solution to overcome these challenges.
Radiolabeled Agents for Molecular Imaging and/or Therapy
Published in George C. Kagadis, Nancy L. Ford, Dimitrios N. Karnabatidis, George K. Loudos, Handbook of Small Animal Imaging, 2018
Dimitrios Psimadas, Eirini A. Fragogeorgi
A strategy that can bypass the drawbacks of radiolabeled antibodies is the radiolabeling of peptides. In general, peptides are small amino acid sequences that have low molecular weight, consist of fewer than 50 amino acids, and regulate many metabolic processes (Reubi 2003). These small peptides are rapidly taken up and retained in the target tissues by coupling to specific receptors, functioning either as agonists or antagonists in specific biological processes. Most agonists exert their function by binding to G-coupled proteins, which results in internalization of the peptide–receptor complex, whereas the majority of antagonists remain on the external side of the cellular membrane (Krohn 2001). These characteristics, together with their rapid plasma clearance due to renal excretion, make them very suitable for imaging. Up to now, their clinical use has been limited to tumor imaging and treatment (mostly neuroendocrine tumors) and to some extent to the diagnosis of infectious and inflammatory diseases. In several tumor types, and to a lesser extent in some inflammatory diseases, many of the peptide receptors are overexpressed, thereby forming a potential target for molecular imaging and therapy (Reubi 2003). The peptide synthesis and development is relatively cheap and can be performed quickly with automated synthesis units. Peptides are not immunogenic, and they usually demonstrate good tumor penetration and present fast tumor uptake, having low bone marrow uptake (Eberle and Mild 2009).
A non-functional neoepitope specific CD8+ T-cell response induced by tumor derived antigen exposure in vivo
Published in OncoImmunology, 2019
Mathias Vormehr, Katharina Reinhard, Renata Blatnik, Kathrin Josef, Jan David Beck, Nadja Salomon, Martin Suchan, Abderraouf Selmi, Fulvia Vascotto, Johannes Zerweck, Holger Wenschuh, Mustafa Diken, Sebastian Kreiter, Özlem Türeci, Angelika B. Riemer, Ugur Sahin
Peptide synthesis was performed by JPT Peptide Technologies GmbH via fully automated SPOT-synthesis approach (PepTrack™ Fast Track specification). A cellulose membrane was functionalized with the individual C-terminal amino acid for each peptide. After Fmoc- deprotection and washing, the activated amino acids were spotted to the membrane in a computer controlled and spatially addressed fashion. Deprotection, washing and amino acid coupling cycles were repeated until the complete peptide sequences were assembled. Following side-chain deprotection peptides were individually cleaved into microtiter-plate wells, analyzed by high-throughput HPLC-MS and dried. Finally, peptides were pooled and aliquoted in antigen representing matrix pools using an automated liquid handling system. RNAs were synthesized by BioNTech RNA Pharmaceuticals GmbH.12 Smc3 RNA encodes 27 amino acids with the mutated amino acid in the center (position 14). PME1 RNA represents five neoepitopes, CT26-ME1 to CT26-ME5 (27 amino acids per epitope) as described earlier.12 gp70 RNA codes for the H-2Ld-restricted epitope AH1423-431 derived from the murine leukemia virus envelope glycoprotein 70 (gp70) with single amino acid substitution at position five (V427A).18 All epitope sequences were embedded in a backbone described by Kreiter and colleagues.24
Cell penetrating peptides: a comparative transport analysis for 474 sequence motifs
Published in Drug Delivery, 2018
Katrin Ramaker, Maik Henkel, Thorsten Krause, Niels Röckendorf, Andreas Frey
All peptides were synthesized by solid phase peptide synthesis under rigorous coupling conditions to minimize truncation of peptides and to ensure that only correct full-length peptides were equipped with the cargo of interest. This included a double coupling procedure with 10-fold excess of fluorenylmethyloxycarbonyl (Fmoc) protected amino acid monomers in each synthesis cycle, with strict capping steps to block any remaining free amino functions after each coupling cycle. In the last step of the synthesis, all 474 peptides were equipped N-terminally with a carboxyfluorescein (FAM) moiety as model cargo. This FAM residue not only represents the cargo but also acts as a reporter group (fluorophore) in the biological assay system. Our synthesis procedure guarantees that only completely synthesized peptides can carry the fluorophore and hence give a fluorescence signal in biological assay systems; truncated sequences are capped during synthesis and therefore lack the carboxyfluorescein moiety. To verify the identity of the synthesized CPPs, 10% of all peptides were randomly selected and checked by mass spectrometry; the expected products were confirmed in all preparations analyzed (Supplementary Table TS2).
Advances in venom peptide drug discovery: where are we at and where are we heading?
Published in Expert Opinion on Drug Discovery, 2021
Taylor B. Smallwood, Richard J. Clark
The chemical synthesis of venom-derived peptides can be challenging, as most are around 30–40 amino acids long and many contain several disulfide bonds. Improvement in synthetic methodology such as native chemical ligation and automated solid phase peptide synthesis, as well as recombinant expression allow for the ability to produce structurally complex peptides. The formation of the correct disulfide isomer within a peptide is crucial for peptide activity. The linkage of a disulfide bond is a oxidation reaction involving the thiol groups of two cysteine residues. A peptide that has multiple cysteine residues within its sequence has the possibility to create multiple disulfide bond isomers. For example, a peptide with four, six, or eight cysteines can potentially form 3, 15, or 105 different disulfide isomers respectively. Incorrect pairing of disulfide bonds can lead to a non-native fold of the peptide and therefore loss of bioactivity. The chemical method for disulfide bond formation in cysteine-rich peptides is typically subjecting the reduced peptide to a freely oxidative folding reaction. This process generally provides the native disulfide bond pairing; however, this needs to be verified as some isomers can still occur. In order to overcome incorrect disulfide bond pairing, an alternative option is available known as a regioselective strategy. This method involves the stepwise formation of disulfide bonds, accomplished using various orthogonally protected cysteine residues [95–97]. The advancement of these methods should allow for the accelerated synthesis of venom-derived peptides in the peptide drug development pipeline.