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Used in solid-phase peptide synthesis (SPPS) for peptides with an acid termination, the 2-chlorotrityl chloride (2-CTC) resin is highly susceptible to moisture, leading to reduced resin loading and lower synthetic yields. It is therefore recommended that the resin be activated with thionyl chloride (SOCl2) before peptide assembly. Here we present an optimized procedure for resin activation that minimizes the use of SOCl2 as the activation reagent and reduces the activation time. Additionally, we demonstrate the feasibility of reusing the 2-CTC resin when following the activation protocol, achieving comparable results to the first usage of the resin. Moreover, we achieved different degrees of resin activation by varying the amount of SOCl2. For instance, the use of 2% SOCl2 in anhydrous dichloromethane (DCM) allowed up to 44% activation of the resin, thereby making it suitable for the synthesis of longer peptides. Alternatively, employing 25% SOCl2 in anhydrous DCM resulted in up to 80% activation with a reaction time of only 5 min in both cases.

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Matrix effect and sample pretreatment significantly affect the percentage recovery of peptides in biological matrices, affecting the method robustness and accuracy. To counteract this effect, an internal standard (IS) is used; however, in most cases this is not available, which limits the analytical method. It is important to identify short peptides that can be used as ISs in the quantification of peptides in biological matrices. In this study, doping peptides GHRP-4, GHRP-5, GHRP-6, Sermorelin (1-11), Sermorelin (13-20) and Sermorelin (22-29) were synthesized using solid-phase peptide synthesis. Treatment with human blood, trypsin and chymotrypsin was used to determine the stability of the peptides. Products were evaluated using the high-performance liquid chromatography-diode array detector (HPLC-DAD) method. The analytical methodology and sample pretreatment were effective for the analysis of these molecules. A unique profile related to protein binding and enzymatic stability of each peptide was established. GHRP-4, GHRP-6 and Sermorelin (22-29) can be considered as in-house ISs as they were stable to enzyme and blood treatment and can be used for the quantification of peptides in biological samples. Peptides GHRP-6 and Sermorelin (22-29) were used to analyse a dimeric peptide (26 [F] LfcinB (20-30)2 ) in four different matrices to test these peptides as in-house IS.

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The interest in therapeutic monoclonal antibodies (mAbs) has significantly grown in the pharmaceutical industry, exceeding 100 FDA mAbs approved. Although the upstream processing of their industrial production has been significantly improved in the last years, the downstream processing still depends on immobilized protein A affinity chromatography. The high cost, low capacity and short half-life of immobilized protein A chromatography matrices, encouraged the design of alternative short-peptide ligands for mAb purification. Most of these peptides have been obtained by screening combinatorial peptide libraries. These low-cost ligands can be easily produced by solid-phase peptide synthesis and can be immobilized on chromatographic supports, thus obtaining matrices with high capacity and selectivity. Furthermore, matrices with immobilized peptide ligands have longer half-life than those with protein A due to the higher stability of the peptides. In this review the design and synthesis of peptide ligands, their immobilization on chromatographic supports and the evaluation of the affinity supports for their application in mAb purification is described.

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In this chapter, a protocol to design affinity chromatography matrices with short peptide ligands immobilized for protein purification is described. The first step consists of the synthesis of a combinatorial peptide library on the hydroxymethylbenzoyl (HMBA)-ChemMatrix resin by the divide-couple-recombine (DCR) method using the Fmoc chemistry. Next, the library is screened with the protein of interest labeled with a fluorescent dye or biotin. Subsequently, peptides contained on positive beads are identified by tandem matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS/MS), and those sequences showing greater consensus are synthesized in larger quantities and immobilized on chromatographic supports. Finally, target protein adsorption on peptide affinity matrices is evaluated through equilibrium adsorption isotherms and breakthrough curves.

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Scorpion and spider envenomation is treated with the appropriate antivenoms, prepared as described by Césaire Auguste Phisalix and Albert Calmette in 1894. Such treatment requires the acquisition and manipulation of arachnid venoms, both very complicated procedures. Most of the toxins in the venoms of spiders and scorpions are extremely stable cysteine-rich peptide neurotoxins. Many strategies have been developed to obtain synthetic immunogens to facilitate the production of antivenoms against these toxins. For example, whole peptide toxins can be synthesized by solid-phase peptide synthesis (SPPS). Also, epitopes of the toxins can be identified and after the chemical synthesis of these peptide epitopes by SPPS, they can be coupled to protein carriers to develop efficient immunogens. Moreover, multiple antigenic peptides with a polylysine core can be designed and synthesized. This review focuses on the strategies developed to obtain synthetic immunogens for the production of antivenoms against the toxic Cys-rich peptides of scorpions and spiders.

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During the final step of t-Boc/Bzl, solid-phase peptide synthesis (SPPS)-protecting groups from amino acids (aa) side chains must be removed from the target peptides during cleavage from the solid support. These reaction steps involve hydrolysis with hydrogen fluoride (HF) in the presence of a nucleophile (scavenger), whose function is to trap the carbocations produced during SN 1-type reactions. Five peptide sequences were synthesised for evaluating p-methoxyphenol effectiveness as a potent scavenger. After the synthesis, the resin-peptide was then separated into two equal parts to be cleaved using two scavengers: conventional reactive p-cresol (reported in the literature as an effective acyl ion eliminator) and p-methoxyphenol (hypothesised as fulfilling the same functions as the routinely used scavenger). Detailed analysis of the electrostatic potential map (EPM) revealed similarities between these two nucleophiles, regarding net atomic charge, electron density distribution, and similar pKa values. Good scavenger efficacy was observed by chromatography and mass spectrometry results for the synthesised molecules, which revealed that p-methoxyphenol can be used as a potent scavenger during SPPS by t-Boc/Bzl strategy, as similar results were obtained using the conventional scavenger.

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Bevacizumab is a monoclonal antibody, produced in CHO cells, used for the treatment of many human cancers. It is an anti-vascular endothelial growth factor (antsi-VEGF) that blocks the growth of tumor blood vessels. Nowadays its purification is achieved by affinity chromatography (AC) using protein A which is a very expensive ligand. On the other hand, the peptide Ac-PHQGQHIGVSK contained in the VEGF fragment binds bevacizumab with high affinity. This short peptide ligand has higher stability and lower cost than protein A and it can be prepared very easily by solid phase peptide synthesis. The present protocol describes the synthesis of Ac-PHQGQHIGVSK-agarose and its use for affinity chromatography purification of bevacizumab from a clarified CHO cell culture. •Ac-PHQGQHIGVSK-agarose capacity and selectivity are equivalent to those of protein A matrices.•The peptide ligand shows a greater stability and lower cost. The lack of Trp, Met or Cys in the peptide ligand prevents its oxidation and extends the useful life of the chromatographic matrix.•Mild conditions used during chromatography preserved the integrity of bevacizumab.

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A methodology was implemented for purifying peptides in one chromatographic run via solid-phase extraction (SPE), reverse phase mode (RP), and gradient elution, obtaining high-purity products with good yields. Crude peptides were analyzed by reverse phase high performance liquid chromatography and a new mathematical model based on its retention time was developed in order to predict the percentage of organic modifier in which the peptide will elute in RP-SPE. This information was used for designing the elution program of each molecule. It was possible to purify peptides with different physicochemical properties, showing that this method is versatile and requires low solvent consumption, making it the least polluting one. Reverse phase-SPE can easily be routinely implemented. It is an alternative to enrich and purified synthetic or natural molecules.

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An 82-residue-long chimeric peptide was synthesised by solid phase peptide synthesis (SPPS), following the Fmoc protocol. Microwave (MW) radiation-assisted synthesis was compared to standard synthesis using low loading (0.20 mmol/g) of polyethylene glycol (PEG) resin. Similar synthetic difficulties were found when the chimeric peptide was obtained via these two reaction conditions, indicating that such difficulties were inherent to the sequence and could not be resolved using MW; by contrast, the number of coupling cycles and total reaction time became reduced whilst crude yield and percentage recovery after purification were higher for MW radiation-assisted synthesis.

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Peptide KVPLITVSKAK was selected to design a synthetic ligand for affinity chromatography purification of recombinant human follicle stimulating hormone (rhFSH), based on the interaction of the hormone with the exoloop 3 of its receptor. The peptide was acetylated to improve its stability to degradation by exopeptidases. A cysteine was incorporated at the C-termini to facilitate its immobilization to the chromatographic activated SulfoLink agarose resin. A sample of crude rhFSH was loaded to the affinity column, using 20 mM sodium phosphate, 0.5 mM methionine, and pH 5.6 and 7.2 as adsorption and elution buffers, respectively. The dynamic capacity of the matrix was 54.6 mg rhFSH/mL matrix and the purity 94%. The percentage of oxidized rhFSH was 3.4%, and that of the free subunits was 1.2%, both in the range established by the European Pharmacopeia, as also were the sialic acid content and the isoforms profile.

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Recombinant human growth hormone (rhGH) is used for the treatment of several pathologies, most of them related to growth. Although different expression systems can be used for its production, the milk from transgenic cows is one of the most interesting due to the high rhGH level achieved (5 g/L). We have designed and synthesized short peptides (9 or 10 amino acid long) using Fmoc chemistry and studied their ability to purify rhGH from milk once immobilized on an agarose support. Using spiked milk with the hormone as a sample, rhGH was purified with 88% yield and 92% purity in a single step with a fold purification of 4.5. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:999-1005, 2018.

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The results from the synthesis of peptides by Fmoc/SPPS on a 2-CTC resin and then lactamization in solution or solid phase for the preparation of cyclopeptides are presented. Both procedures allow the synthesis of the desired compounds in good to very good yield and with high cyclization efficiency for on-resin macrocyclization. In addition, the activities of the corresponding cyclopeptides against the chloroquine-resistant K1 strain of Plasmodium falciparum were evaluated. Cyclo-Cys(Trt)-Gly-Thr( tBu)-Gly-Cys(Trt)-Gly showed potent in vitro and selective activity against this parasite, EC50 = 28 nM.

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The deprotection step is crucial in order to secure a good quality product in Fmoc solid phase peptide synthesis. 9-Fluorenylmethoxycarbonyl (Fmoc) removal is achieved by a two-step mechanism reaction favored by the use of cyclic secondary amines; however, the efficiency of the reaction could be affected by side reactions and by-product formation. Several aspects have to be taken into consideration when selecting a deprotection reagent: its physicochemical behavior, basicity (pKa) and polarity, concentration, and time of reaction, toxicity and disposability of residues and, finally, availability of reagents. This report presents a comparison of the performance of three strategies for deprotection using microwave-assisted Fmoc peptide synthesis. Four peptide sequences were synthesized using Rink amide resin with a Liberty Blue™ automated synthesizer and 4-methylpiperidine (4MP), piperidine (PP), and piperazine (PZ) as Fmoc removal reagents. In the first instance all three reagents behaved similarly. A detailed analysis showed a correlation between the hydrophobicity and size of the peptide with the yield and purity of the obtained product. The three reagents are interchangeable, and replacement of piperidine could be advantageous regarding toxicity and reagent handling.

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Many amyloidogenic peptides are highly hydrophobic, introducing significant challenges to obtaining high quality peptides by chemical synthesis. For example, while good yield and purity can be obtained in the solid-phase synthesis of the Alzheimer's plaque peptide Aß40, addition of a C-terminal Ile-Ala sequence to generate the more toxic Aß42 molecule creates a much more difficult synthesis resulting in low yields and purities. We describe here a new method that significantly improves the Fmoc solid-phase synthesis of Aß peptides. In our method, Lys residues are linked to the desired peptide's C-terminus through standard peptide bonds during the synthesis. These Lys residues are then removed post-purification using immobilized carboxypeptidase B (CPB). With this method we obtained both Aß42 and Aß46 of superior quality that, for Aß42, rivals that obtained by recombinant expression. Intriguingly, the method appears to provide independent beneficial effects on both the total synthetic yield and on purification yield and final purity. Reversible Lys addition with CPB removal should be a generally useful method for making hydrophobic peptides that is applicable to any sequence not ending in Arg or Lys. As expected from the additional hydrophobicity of Aß46, which is extended from the sequence Aß42 by a C-terminal Thr-Val-Ile-Val sequence, this peptide makes typical amyloid at rates significantly faster than for Aß42 or Aß40. The enhanced amyloidogenicity of Aß46 suggests that, even though it is present in relatively low amounts in the human brain, it could play a significant role in helping to initiate Aß amyloid formation.

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