TDPBP Derivative Aiding Liquid-Phase Synthesis Strategy and ACE Inhibitory Structure-activity Relationship of Anti-SARS Octapeptide

The tri(4'-diphenylphosphonyloxylbenzoyl phenyl) phosphate (TDPBP) derivatives were designed and developed as C-terminal supports to aid the greener and highefficient liquid-phase peptide synthesis (LPPS) without the need of unrecyclable resin and chromatographic separation, whereby the anti-SARS octapeptide (2) (AVLQSGFR) was synthesized with TDPBP-OH support via Fmoc chemistry and support-aided precipitation (SAP) technology. Furthermore, the ACE inhibition and the inhibitory structure-activity relationship (SAR) between the synthetic C-terminal amidated derivate (1), anti-SARS octapeptide (2) and its alanine-scanning sequence analogues (3) to (9) were systematically studied by HPLC analysis and 3D-QSAR via molecular docking.<br>

TDPBP Derivative Aiding Liquid-Phase Synthesis Strategy and ACE Inhibitory Structure-activity Relationship of Anti-SARS Octapeptide

The tri(4'-diphenylphosphonyloxylbenzoyl phenyl) phosphate (TDPBP) derivatives were designed and developed as C-terminal supports to aid the greener and highefficient liquid-phase peptide synthesis (LPPS) without the need of unrecyclable resin and chromatographic separation, whereby the anti-SARS octapeptide (2) (AVLQSGFR) was synthesized with TDPBP-OH support via Fmoc chemistry and support-aided precipitation (SAP) technology. Furthermore, the ACE inhibition and the inhibitory structure-activity relationship (SAR) between the synthetic C-terminal amidated derivate (1), anti-SARS octapeptide (2) and its alanine-scanning sequence analogues (3) to (9) were systematically studied by HPLC analysis and 3D-QSAR via molecular docking.<br>

Synthesis of Phosphorodiamidate Morpholino Oligonucleotides Using Trityl and Fmoc Chemistry-A New Method Amenable to Automated Synthesizer

<p>Phosphorodiamidatemorpholino oligonucleotides (PMO) are routinely used for gene silencing and the recently developed PMO-based drug “Exondys51” has highlighted the importance of PMO as excellent antisense reagents. However, the synthesis of PMO has remained challenging. Here a method for the synthesis of PMO using either trityl or Fmoc-protected active morpholino monomers using chlorophosphoramidate chemistry in the presence of a suitable coupling agent on a solid support has been reported. After screening several coupling agents (tetrazole, 1,2,4-triazole, ETT, iodine, LiBr and dicyanoimidazole), ETT and iodine were found to be suitable for efficient coupling. Fmoc chemistry was not known for PMO synthesis because the preparation of Fmoc-protected chlorophosphoramidate monomers was not trivial. Synthesis of Fmoc-protected activated monomers and their use in PMO synthesis is reported for the first time. 25-mer PMO has been synthesized using both the methods and validated <i>in vivo</i> in the zebrafish model by targeting the <i>no tail</i> gene. Methods have been transferred in DNA synthesizer which has become user friendly for PMO synthesis and opened a new avenue to explore PMO for various applications. Fmoc chemistry could be suitable for scalable approach of PMO synthesis using peptide synthesizer as it is a neutral oligomer like peptide.</p>

Synthesis of Phosphorodiamidate Morpholino Oligonucleotides Using Trityl and Fmoc Chemistry-A New Method Amenable to Automated Synthesizer

<p>Phosphorodiamidatemorpholino oligonucleotides (PMO) are routinely used for gene silencing and the recently developed PMO-based drug “Exondys51” has highlighted the importance of PMO as excellent antisense reagents. However, the synthesis of PMO has remained challenging. Here a method for the synthesis of PMO using either trityl or Fmoc-protected active morpholino monomers using chlorophosphoramidate chemistry in the presence of a suitable coupling agent on a solid support has been reported. After screening several coupling agents (tetrazole, 1,2,4-triazole, ETT, iodine, LiBr and dicyanoimidazole), ETT and iodine were found to be suitable for efficient coupling. Fmoc chemistry was not known for PMO synthesis because the preparation of Fmoc-protected chlorophosphoramidate monomers was not trivial. Synthesis of Fmoc-protected activated monomers and their use in PMO synthesis is reported for the first time. 25-mer PMO has been synthesized using both the methods and validated <i>in vivo</i> in the zebrafish model by targeting the <i>no tail</i> gene. Methods have been transferred in DNA synthesizer which has become user friendly for PMO synthesis and opened a new avenue to explore PMO for various applications. Fmoc chemistry could be suitable for scalable approach of PMO synthesis using peptide synthesizer as it is a neutral oligomer like peptide.</p>

Synthesis of piperidine-4-one Derivative Containing Dipeptide: An Acetyl cholinesterase and β-secretase Inhibitor

Background: With the goal of developing Alzheimer's disease therapeutics, we have designed and synthesized novel piperidone fused dipeptide (DPPS) derivatives possessing dual action such as acetylcholinesterase (AChE) and beta-amyloid peptide (Aβ) aggregation inhibition. Designed peptide was synthesized by solid phase peptide synthesis using FMOC chemistry protocol and characterized by mass spectroscopy. Methods: The amino acid sequence in peptide was analyzed by LC-MS-MS. In silico docking analysis was carried out using GLIDE software. The docking score using GLIDE was found to be -7.88 against AChE and -9.74 against BACE1 enzyme. In vitro enzyme inhibition assay was carried out for AChE enzyme and BACE1 enzyme. Results: The IC50 values of AChE inhibition and BACE1 of DPPS were found to be 0.4796 μM/ml and 0.0154 μM/ml, respectively. The correlation of in silico and in vitro results showed that DPPS possessed a greater ability to inhibit BACE1 enzyme.

Cyclic Tripeptide-based Potent and Selective Human SIRT5 Inhibitors

Background:: SIRT5 is one of the seven members (SIRT1-7) of the mammalian sirtuin family of protein acyl-lysine deacylase enzymes. In recent years, important regulatory roles of SIRT5 in (patho)physiological conditions (e.g. metabolism and cancer) have been increasingly demonstrated. For a better biological understanding and therapeutic exploitation of the SIRT5- catalyzed deacylation reaction, more effort on identifying potent and selective SIRT5 inhibitors beyond those currently known would be rewarding. Objective:: In the current study, we would like to see if it would be possible to develop potent and selective SIRT5 inhibitory lead compounds with a novel structural scaffold than those of the currently known potent and selective SIRT5 inhibitors. Methods: : In the current study, six N-terminus-to-side chain cyclic tripeptides (i.e. 8-13) each harboring the thiourea-type catalytic mechanism-based SIRT5 inhibitory warhead Nε-carboxyethylthiocarbamoyl- lysine as the central residue were designed, synthesized by the Nα-9- fluorenylmethoxycarbonyl (Fmoc) chemistry-based solid phase peptide synthesis (SPPS) on the Rink amide 4-methylbenzhydrylamine (MBHA) resin, purified by the semi-preparative reversedphase high performance liquid chromatography (RP-HPLC), characterized by the high-resolution mass spectrometry (HRMS); and were evaluated by the in vitro sirtuin inhibition assay and the in vitro proteolysis assay. Results:: Among the cyclic tripeptides 8-13, we found that 10 exhibited a potent (IC50 ~2.2 μM) and selective (≥60-fold over the SIRT1/2/3/6-catalyzed deacylation reactions) inhibition against the SIRT5-catalyzed desuccinylation reaction. Moreover, 10 was found to exhibit a ~42.3-fold stronger SIRT5 inhibition and a greater proteolytic stability than its linear counterpart 14. Conclusion:: With a novel and modular structural scaffold as compared with those of all the currently reported potent and selective SIRT5 inhibitors, 10 could be also a useful and feasible lead compound for the quest for superior SIRT5 inhibitors as potential chemical/pharmacological probes of SIRT5 and therapeutics for human diseases in which SIRT5 desuccinylase activity is upregulated.

Antibacterial Activities of Lipopeptide (C10)2-KKKK-NH2 Applied Alone and in Combination with Lens Liquids to Fight Biofilms Formed on Polystyrene Surfaces and Contact Lenses

The widespread use of biomaterials such as contact lenses is associated with the development of biofilm-related infections which are very difficult to manage with standard therapies. The formation of bacterial biofilms on the surface of biomaterials is associated with increased antibiotic resistance. Owing to their promising antimicrobial potential, lipopeptides are being intensively investigated as novel antimicrobials. However, due to the relatively high toxicity exhibited by numerous compounds, a lot of attention is being paid to designing new lipopeptides with optimal biological activities. The principal aim of this study was to evaluate the potential ophthalmic application of lipopeptide (C10)2-KKKK-NH2. This lipopeptide was synthesized according to Fmoc chemistry using the solid-phase method. The antibiofilm activities of the lipopeptide, antibiotics used in ocular infections, and commercially available lens liquids were determined using the broth dilution method on polystyrene 96-well plates and contact lenses. Resazurin was applied as the cell-viability reagent. The effectiveness of the commercially available lens liquids supplemented with the lipopeptide was evaluated using the same method and materials. (C10)2-KKKK-NH2 exhibited stronger anti-biofilm properties compared to those of the tested conventional antimicrobials and showed the ability to enhance the activity of lens liquids at relatively low concentrations (4–32 mg/L). Estimation of the eye irritation potential of the lipopeptide using Toxtree software 2.6.13 suggests that the compound could be safely applied on the human eye. The results of performed experiments encourage further studies on (C10)2-KKKK-NH2 and its potential application in the prophylaxis of contact lens-related eye infections.

Identification of the primary peptide inhibitor contaminant of fibrillation and toxicity in synthetic Amyloid-β42

Understanding the pathophysiology of Alzheimer disease has relied upon the use of amyloid peptides from a variety of sources, but most predominantly synthetic peptides produced using t-butyloxycarbonyl (Boc) or 9-fluorenylmethoxycarbonyl (Fmoc) chemistry. These synthetic methods can lead to minor impurities which can have profound effects on the biological activity of amyloid peptides. Here we used a combination of cytotoxicity assays, fibrillation assays and high resolution mass spectrometry (MS) to identify impurities in synthetic amyloid preparations that inhibit both cytotoxicity and aggregation. We identify the Aβ42Δ39 species as the major peptide contaminant responsible for limiting both cytotoxicity and fibrillation of the amyloid peptide. In addition, we demonstrate that the presence of this minor impurity inhibits the formation of a stable Aβ42 dimer observable by MS in very pure peptide samples. These results highlight the critical importance of purity and provenance of amyloid peptides in Alzheimer’s research in particular, and biological research in general.

Synthetic Peptides as Potential Antigens for Cutaneous Leishmaniosis Diagnosis

This work’s goal was to research new candidate antigens for cutaneous leishmaniosis (CL). In order to reach the goal, we used random peptide phage display libraries screened using antibodies fromLeishmania braziliensispatients. After selection, three peptides (P1, P2, and P3) were synthesized using Fmoc chemistry. The peptides individually or a mixture of them (MIX) was subsequently emulsified in complete and incomplete Freund’s adjuvant and injected subcutaneously in golden hamsters. Sera from the hamsters administered with P1 presented antibodies that recognized proteins between 76 and 150 kDa fromL. braziliensis. Sera from hamsters which had peptides P2 and P3, as well as the MIX, administered presented antibodies that recognized proteins between 52 and 76 kDa ofL. braziliensis. The research on the similarity of the peptides’ sequences in protein databases showed that they match a 63 kDa glycoprotein. The three peptides and the MIX were recognized by the sera from CL patients by immunoassay approach (ELISA). The peptides’ MIX showed the best performance (79% sensitivity) followed by the P1 (72% sensitivity), and the AS presented 91% sensitivity. These results show a new route for discovering molecules for diagnosis or for immunoprotection against leishmaniosis.