Synthesis of Dehydrodipeptide and N-ethyl-dehydrodipeptide Derivatives with an α-Aminoisobutyric Acid Residue

Several dipeptides with an N-benzyloxycarbonyl or an N-(tert-butyloxycarbonyl) α-aminoisobutyric acid residue and a β-hydroxyamino acid methyl ester were subjected to dehydration to form dehydrodipeptide derivatives. N-Ethylation of these dipeptides using triethyloxonium tetrafluoroborate with potassium tert-butoxide as auxiliary base led to complex mixtures that failed to render pure products. However, when the 4-nitrophenylsulfonyl protecting group was substituted for the benzyloxycarbonyl or tert-butyloxycarbonyl groups and N,N-diisopropylethylamine was substituted for potassium tert-butoxide, the dehydrodipeptide derivatives were selectively N-alkylated at the amino terminal nitrogen in fair to high yields. Alternatively, N-ethylation can be carried out prior to dehydration. Thus, through a combination of dehydration and N-ethylation procedures, it was possible to obtain dipeptides with α-aminoisobutyric acid and dehydroamino acid residues, which are N-alkylated at the amino terminal nitrogen.Foundation for Science and Technology (FCT) – Portugal and Fundo Europeu de Desenvolvimento Regional (FEDER) for financial support to Chemistry Centre of University of Minho. The NMR spectrometer Bruker Avance II+ 400 is part of the National NMR Network and was purchased in the framework of the National Program for Scientific Re-equipment; contract REDE/1517/RMN/2005, with funds from POCI 2010, FEDER and FCT.info:eu-repo/semantics/publishedVersio

Synthesis of N-alkyl-C α, α-dimethylglycine derivatives

The application of trialkyloxonium tetrafluoroborates for N-alkylation of the nonnatural amino acid Cα, α-dimethylglycine is described. Several methyl esters of dimethylglycine protected with different amine protecting groups were subject to N-ethylation or N-methylation with triethyloxonium tetrafluoroborate or trimethyloxonium tetrafluoroborate, respectively. The corresponding N-akyl-Cα, α-dimethylglycine derivatives were obtained in good to high yields. Removal of the methyl ester rendered amino acid derivatives ready for application in peptide synthesis.Foundation for Science and Technology (FCT) – Portugal and Fundo Europeu de Desenvolvimento Regional (FEDER) for financial support to Chemistry Centre of University of Minho. The NMR spectrometer Bruker Avance II+ 400 is part of the National NMR Network and was purchased in the framework of the National Program for Scientific Re-equipment; contract REDE/1517/RMN/2005, with funds from POCI 2010, FEDER and FCT.info:eu-repo/semantics/publishedVersio

Synthesis of Peptaibolin, an antimicrobial peptide

To tackle one of the biggest global health problems, the resistance of microorganisms to antibiotics, a collective effort in the search for more effective agents against bacteria was required. Peptides with antimicrobial activity have been raising much attention as a promising alternative for antibiotics. Peptaibols, for instance, are a family of antimicrobial peptides (AMPs) with great biomedical potential, in which the Peptaibolin can be highlighted. This peptide has gained relevance due to its small amino acids content, only four, and its acetyl group and a phenylalaninol residue (Phol) at the N-terminal and C-terminal, respectively. Here, we report the synthesis of Peptaibolin through Solid Phase Peptide Synthesis assisted by Microwave heating (MW-SPPS) in a pre-loaded Phe-Wang resin. Starting from a loading of 0.51 mmol/g, two syntheses were made, using two different combinations of coupling reagents. The best option was DIC/Oxima, achieving a yield of 50.0%. Proton Nuclear Magnetic Resonance (1H-NMR) studies confirmed the peptide structure, while High Performance Liquid Chromatography (HPLC) verified the peptide purity. The peptide solubility was examined against several combinations of solvents. Peptaibolin was not soluble in water, only in organic solvents or in the combination of both. Antimicrobial testing was conducted using Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, and Pseudomonas aeruginosa. Minimum inhibitory concentration studies demonstrated the resistance of bacteria to the peptide action and the peptide instability in bacterial growth conditionsFundação para a Ciência e Tecnologia-FCT (Portugal) for funding through CQUM (UID/QUI/00686/2020) and project PTDC/QUI-COL/28052/2017. The NMR spectrometer Bruker Avance III 400 is part of the National NMR Network and was purchased within the framework of the National Program for Scientific Re-equipment, contract REDE/1517/RMN/2005 with funds from POCI 2010 (FEDER) and FCT. Authors also acknowledge FCT for funding the project PEPTEX with reference PTDC/CTM-TEX/28074/2017 (POCI-01-0145-FEDER-028074). They acknowledge project UID/CTM/00264/2021 of Centre for Textile Science and Technology (2C2T), funded by national funds through FCT/MCTE

Deri yara kaplamalarindaki uygulamalar için elektrik alan lif çekim ile üretilmiş Tiger 17 peptit yüklü polikaprolakton/selüloz asetat nano lifli matlar

A skin wound if not properly treated can result in a chronic wound susceptible to widespread infections, which can result in the patient's death. Currently, tissue engineering is described as an interdisciplinary field that combines principles of engineering, chemistry and biology to generate solutions that allow to repair, restore and/or improve the functions of injured tissues. In the same sense, the textile area addresses solutions based on polymeric fibers, produced from a wide range of polymers, which allow the generation of structures with a large surface area, porosity and mechanical resistance that can be used as bioactive dressings that promote a healing and efficient antimicrobial activity. This research work focused on the synthesis of Tiger 17, through microwave-assisted solid-phase synthesis methodologies, and Tiger 17 commercially obtained, respective structural characterization and evaluation of the antimicrobial capacity. Simultaneously, nanofibrous polymer matrices were produced using the electrospinning technique with the aim of immobilizing the developed biomolecule and thus creating potential vehicles for a local and sustainable antimicrobial action (controlled release). In order to verify its physical and chemical properties, advanced characterization techniques were used: proton nuclear magnetic resonance, high performance liquid chromatography, optical microscopy, scanning electron microscopy, fourier transform infrared spectroscopy–attenuated total reflectance, thermogravimetry, differential scanning calorimetry, contact angle and surface energy and determination of porosity and hydration.Düzgün tedavi edilmeyen bir cilt yarası, hastanın ölümüyle sonuçlanabilecek yaygın enfeksiyonlara duyarlı kronik bir yaraya neden olabilir. Günümüzde doku mühendisliği, hasarlı dokuların işlevlerini onarmaya, eski haline getirmeye ve/veya iyileştirmeye olanak tanıyan çözümler üretmek için mühendislik, kimya ve biyolojinin ilkelerini birleştiren disiplinler arası bir alan olarak tanımlanmaktadır. Bu kapsamda, tekstil, iyileşmeyi ve etkili antimikrobiyal aktiviteyi teşvik eden biyoaktif pansuman olarak kullanılabilecek, geniş yüzey alanlı, gözenekli ve mekanik dirençli yapıların oluşturulmasına izin veren, geniş bir polimer yelpazesinden üretilen liflere dayalı çözümler sunmaktadır. Bu araştırma çalışması, mikrodalga destekli katı faz sentez metodolojileri aracılığıyla Tiger 17'nin sentezine ve ticari olarak temin edilen Tiger 17 ile birlikte yapısal karakterizasyonunun ve antimikrobiyal kapasitenin değerlendirilmesine odaklanmıştır. Aynı zamanda, geliştirilen biyomolekülü hareketsiz hale getirmek ve böylece lokal ve sürdürülebilir bir antimikrobiyal etki (kontrollü salım) için potansiyel çözümler yaratmak amacıyla elektrik alan lif üretim tekniği kullanılarak nanolifli polimer matrisler üretildi. Yapının fiziksel ve kimyasal özelliklerini doğrulamak için, proton nükleer manyetik rezonans, yüksek performanslı sıvı kromatografisi, optik mikroskopi, taramalı elektron mikroskobu, fourier dönüşümü kızılötesi spektroskopisi-zayıflatılmış toplam yansıma, termogravimetri, diferansiyel taramalı kalorimetri, temas açısı, yüzey enerjisi, gözeneklilik ve su tutma ölçümleri gibi ileri karakterizasyon teknikleri kullanıldı

Peptide-induced permeation of model membranes by antimicrobial peptidomimetics

In the present communication, a set of Peptaibolin and several peptidomimetics incorporating unnatural α,α-dialkylglycines (Deg, Dpg, Ac6c) were studied for their ability to interact and permeate model membranes from phosphatidylcholine/cholesterol, in different ratios. The permeation activity was monitored by fluorescence spectroscopy, following the release of encapsulated 6-carboxyfluorescein.Fundação para a Ciência e a Tecnologia (Portugal) and FEDER-COMPETE-QREN-EU for financial support through projects PTDC/QUI-BIQ/118389/2010 (FCOMP-01-0124-FEDER-020906), PEst-C/QUI/UI0686/2013 (F-COMP-01-0124-FEDER-037302), and PEst-C/FIS/UI0607/2013 (F-COMP-01-0124-FEDER-022711)

Permeation of model membranes by Peptaibolin mimetics bearing different α,α-dialkylglycines

Comunicação oral OC19 no XV EPI- Iberian Peptide Meeting, Porto, Portugal, 10-12 Fevereiro 2016.It is reported the membrane permeation studies of a model peptide, Peptaibolin (Ac-Leu-Aib-Leu-Aib-Phol), which is the shortest member of the peptaibols family, and several mimetics incorporating unnatural α,α- dialkylglycines (Deg, Dpg, Ac6c) at the native Aib positions. The model membranes were based on small unilamellar vesicles composed of phosphatidylcholines (egg lecithin, DPPC), phosphatidylglycerols (DPPG, DOPG) and cholesterol, at different compositions and ratios, containing a fluorescent probe encapsulated in their aqueous interior, in order to monitor the permeation process by fluorescence spectroscopy. The obtained results revealed a correlation between the length and bulk of the side chain of the unnatural α,α-dialkylglycines and the ability of the corresponding peptide to permeate the model membranes.The authors acknowledge Fundação para a Ciência e a Tecnologia (Portugal) and FEDER-COMPETE-QREN-EU for financial support through projects PTDC/QUIBIQ/118389/2010 (FCOMP-01-0124-FEDER-020906), PEst-C/QUI/UI0686/2013 (F-COMP-01- 0124-FEDER-037302), and PEst-C/FIS/UI0607/2013 (F-COMP-01-0124-FEDER-022711).info:eu-repo/semantics/publishedVersio

Membrane active peptides with unnatural amino acids: permeation studies in model membranes

Comunicação em painel P60 no 2nd Symposium on Medicinal Chemistry, Braga, Portugal, 8 Maio 2015It is reported the membrane permeation studies of Peptaibolin and several mimetics incorporating unnatural α,α-dialkylglycines (Deg, Dpg, Ac6c) with model membranes (egg phosphatidylcholine/cholesterol, in different ratios). The permeation activity was monitored by fluorescence spectroscopy, following release of an encapsulated fluorescent probe (6-carboxyfluorescein). The obtained results revealed a correlation between the length and bulk of the side chain of the unnatural α,α-dialkylglycines and the ability of the corresponding peptide to permeate the model membranes.Fundação para a Ciência e Tecnologia(Portugal) and FEDER-COMPETE for financial support through project PTDC/QUI-BIQ/118389/2010 (FCOMP- 01-0124-FEDER-020906) and PEst-C/QUI/UI0686/2013 (F-COMP-01-0124-FEDER-037302). The NMR spectrometer Bruker Avance III 400 is part of the National NMR Network and was purchased with fund s from FCT and FEDERinfo:eu-repo/semantics/publishedVersio

Self-assembled nanoparticles made of fucan

Amphiphilic polymers can self-assemble in water due to hydrophilic and hydrophobic interactions, forming nanoparticles (NPs) with unique physicochemical characteristics and thermodynamic stability. A non toxic sulfated Fucan, extracted from Spatoglossum schroederi was chemically modified by the grafting of Hexadecylamine (C16) to the polymer hydrophilic backbone. The resulting modified material (Fucan-C16) formed nanosized particles which were characterized by 1H NMR to assess the substitution degree of the hydrophobic chains, fluorescence spectroscopy to determine the critical aggregation concentration (cac), cryo-field emission scanning electron microscopy (Cryo- FESEM) to evaluate the shape and size of the NPs, and dynamic light scattering (DLS) to verify the size distribution. The (cac) of Fucan-C16 NPs ranged between 0.05 and 0.03mg/mL. Cryo-FESEM revealed that Fucan-C16 formed spherical macromolecular particles with diameters between 120 and 180 nm, which were confirmed by DLS. In addition, the size of the NPs were not affected by the concentration of the polymer or by the variation of the pH.The size of nanoparticles increases with increasing its concentration in solution.CAPES, FCT and CNP

Sodium alginate/polycaprolactone co-axial wet-spun microfibers modified with N-carboxymethyl chitosan and the peptide AAPV for Staphylococcus aureus and human neutrophil elastase inhibition in potential chronic wound scenarios

In chronic wound (CW) scenarios, Staphylococcus aureus-induced infections are very prevalent. This leads to abnormal inflammatory processes, in which proteolytic enzymes, such as human neutrophil elastase (HNE), become highly expressed. Alanine-Alanine-Proline-Valine (AAPV) is an antimicrobial tetrapeptide capable of suppressing the HNE activity, restoring its expression to standard rates. Here, we proposed the incorporation of the peptide AAPV within an innovative co-axial drug delivery system, in which the peptide liberation was controlled by N-carboxymethyl chitosan (NCMC) solubilization, a pH-sensitive antimicrobial polymer effective against Staphylococcus aureus. The microfibers' core was composed of polycaprolactone (PCL), a mechanically resilient polymer, and AAPV, while the shell was made of the highly hydrated and absorbent sodium alginate (SA) and NCMC, responsive to neutral-basic pH (characteristic of CW). NCMC was loaded at twice its minimum bactericidal concentration (6.144 mg/mL) against S. aureus, while AAPV was loaded at its maximum inhibitory concentration against HNE (50 μg/mL), and the production of fibers with a core-shell structure, in which all components could be detected (directly or indirectly), was confirmed. Core-shell fibers were characterized as flexible and mechanically resilient, and structurally stable after 28-days of immersion in physiological-like environments. Time-kill kinetics evaluations revealed the effective action of NCMC against S. aureus, while elastase inhibitory activity examinations proved the ability of AAPV to reduce HNE levels. Cell biology testing confirmed the safety of the engineered fiber system for human tissue contact, with fibroblast-like cells and human keratinocytes maintaining their morphology while in contact with the produced fibers. Data confirmed the engineered drug delivery platform as potentially effective for applications in CW care.Authors acknowledge the Portuguese Foundation for Science and Technology (FCT), FEDER funds by means of Portugal 2020 Competitive Factors Operational Program (POCI) and the Portuguese Government (OE) for funding the project PEPTEX with reference PTDC/CTMTEX/28074/2017 (POCI-01-0145-FEDER-028074). Authors also acknowledge project UIDP/00264/2020 of 2C2T and UID/QUI/00686/2020 of CQ, funded by national funds through FCT/MCTES. C.S.M. and H.P.F. also acknowledge FCT for PhD funding via scholarship 2020.08547.BD and for auxiliary researcher contract via 2021.02720.CEEIND, respectively

The Combination of Gefitinib With ATRA and ATO Induces Myeloid Differentiation in Acute Promyelocytic Leukemia Resistant Cells

In approximately 15% of patients with acute myeloid leukemia (AML), total and phosphorylated EGFR proteins have been reported to be increased compared to healthy CD34(+) samples. However, it is unclear if this subset of patients would benefit from EGFR signaling pharmacological inhibition. Pre-clinical studies on AML cells provided evidence on the pro-differentiation benefits of EGFR inhibitors when combined with ATRA or ATO in vitro. Despite the success of ATRA and ATO in the treatment of patients with acute promyelocytic leukemia (APL), therapy-associated resistance is observed in 5-10% of the cases, pointing to a clear need for new therapeutic strategies for those patients. In this context, the functional role of EGFR tyrosine-kinase inhibitors has never been evaluated in APL. Here, we investigated the EGFR pathway in primary samples along with functional in vitro and in vivo studies using several APL models. We observed that total and phosphorylated EGFR (Tyr992) was expressed in 28% and 19% of blast cells from APL patients, respectively, but not in healthy CD34(+) samples. Interestingly, the expression of the EGF was lower in APL plasma samples than in healthy controls. The EGFR ligand AREG was detected in 29% of APL patients at diagnosis, but not in control samples. In vitro, treatment with the EGFR inhibitor gefitinib (ZD1839) reduced cell proliferation and survival of NB4 (ATRA-sensitive) and NB4-R2 (ATRA-resistant) cells. Moreover, the combination of gefitinib with ATRA and ATO promoted myeloid cell differentiation in ATRA- and ATO-resistant APL cells. In vivo, the combination of gefitinib and ATRA prolonged survival compared to gefitinib- or vehicle-treated leukemic mice in a syngeneic transplantation model, while the gain in survival did not reach statistical difference compared to treatment with ATRA alone. Our results suggest that gefitinib is a potential adjuvant agent that can mitigate ATRA and ATO resistance in APL cells. Therefore, our data indicate that repurposing FDA-approved tyrosine-kinase inhibitors could provide new perspectives into combination therapy to overcome drug resistance in APL patients