Search

Several factors have influenced the increasing presence of peptides as an important class of Active Pharmaceutical Ingredients. One is the continued development of synthetic methodologies for peptide synthesis. Herein, we investigated the Fmoc removal step, using the tea-bag strategy. In this regard, three different secondary amines: piperidine, 4-methylpiperidine, and piperazine, were evaluated. As a result of this study, 4-methyl piperidine showed to be an excellent alternative to the usually used piperidine in terms of purity and compliance with green chemistry principles as well., Work funded by Fondecyt, Chilean Grant 1170379.

During the early developmental stage of salmonids, high mortality occurs largely as a result of pathogens. These cause low immune competence in fry, producing disease, decreasing production and finally leading to economic losses. Therefore, the aim of this study was to characterise the developmental stages in which rainbow trout acquires immune response capability when challenged with LPS from Pseudomona aeruginosa for 8 h, studying the hepcidin, cathelicidin-1 and IL-8. Total RNA was extracted from fry at 34, 42, 56 and 66 days post hatching (dph). Hepcidin and cathelicidin-1 transcripts were detected only at days 34 and 42, whereas the IL-8 transcript was detected from day 34 to day 66. To analyse the protein expression in the fry, polyclonal anti-peptide antibodies were generated in rabbit. These three immune sera demonstrated the ability to recognise the whole molecule in biological samples. Immunofluorescence showed that skin, gills and intestine mainly responded to the LPS challenge, indicating that these portals of pathogen entry are capturing LPS. This study constitutes a valuable approach, since it has the potential to identify molecules with biological activity that can be used to evaluate the status of fry in culture.

Cyclotides are circular peptides found in various plant families. A cyclized backbone, together with multiple disulfide bonds, confers the peptides’ exceptional stability against protease digestion and thermal denaturation. In addition, the features of these antimicrobial molecules make them suitable for use in animal farming, such as aquaculture. Fmoc solid phase peptide synthesis on 2-chlorotrityl chlorine (CTC) resin using the “tea-bag” approach was conducted to generate the VarvA cyclotide identified previously from Viola arvensis. MALDI-TOF mass spectrometry determined the correct peptide amino acid sequence and the cyclization sites-critical in this multicyclic compound. The cyclotide showed antimicrobial activity against various Gram-negative bacteria, including recurrent pathogens present in Chilean aquaculture. The highest antimicrobial activity was found to be against Flavobacterium psychrophilum. In addition, membrane blebbing on the bacterial surface after exposure to the cyclotide was visualized by SEM microscopy and the Sytox Green permeabilization assay showed the ability to disrupt the bacterial membrane. We postulate that this compound can be proposed for the control of fish farming infections.

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 BlueTM 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.