We present a systematic study on the best conditions to achieve the microwave-assisted solid phase peptide synthesis of a selected peptaibol, namely the pentapeptide Peptaibolin (Ac-Leu-Aib-Leu-Aib-Phol), by using several resins and different coupling and cleavage conditions aiming at a future synthesis of mimetics of larger members of the peptaibol family bearing bulkier unnatural α,α-dialkylglycines.

We show distinct CH-pi interactions and assembly pathways for the amphiphile N-(fluorenylmethoxycarbonyl)-galactosamine and its epimer N-(fluorenylmethoxycarbonyl)-glucosamine. These differences result in the formation of supramolecular nanofibrous systems with opposite chirality. Our results showcase the importance of the carbohydrates structural diversity for their specific biointeractions and the opportunity that their ample interactome offers for synthesis of versatile and tunable supramolecular (bio)materials.

This work presents our study of optimization of P-HPLC conditions to obtain pure peptaibol mimetics, to be used in biological activity studies. After purification, the sequence and structure of the peptides obtained were confirmed by NMR spectroscopy (1H, 13C, DEPT, HMBC and HMQC spectra) and mass spectrometry.

We report our research on the synthesis of the shortest Peptaibol, Peptaibolin (Ac-Leu-Aib-Leu-Aib-Phol) and several mimetics incorporating unnatural α,α-dialkylglycines at the Aib positions, using microwave-assisted solid phase peptide synthesis performed on a CEM discover SPS device.

A resistência dos micro-organismos à ação dos antibióticos clássicos é um problema sério que afeta a população de todo o planeta, constituindo o alicerce do interesse da comunidade científica pelo desenho e síntese de novos agentes terapêuticos que possam revelar-se uma alternativa aos fármacos atualmente utilizados no tratamento de infeções. Uma das áreas em desenvolvimento é a dos péptidos com atividade antibiótica (AMPs), cuja atividade é mediada pela perturbação da permeabilidade das membranas celulares dos organismos patogénicos. Existem vários tipos de AMPs e entre eles podemos encontrar os peptaibols, uma família de péptidos naturais, isolados de fungos, que apresentam na sua composição α,α-dialquilglicinas. Estes aminoácidos são tetrassubstituídos no átomo de carbono central e restringem a rotação em torno das ligações peptídicas, permitindo obter péptidos com conformações mais definidas e mais resistentes à biodegradação. O alvo de estudo deste trabalho foi o Peptaibolin (péptido mais pequeno da família dos peptaibols), tendo sido sintetizado bem como e vários análogos deste, contendo outras α,α-dialquilglicinas nas posições da α,α-dialquilglicina nativa (o Aib). Para a síntese dos péptidos, com estes aminoácidos estereoquimicamente impedidos, foi usada a estratégia de síntese peptídica em fase sólida assistida por micro-ondas, que tem demostrando resultados muito bons na introdução de aminoácidos difíceis nas sequências peptídicas. As condições usadas na síntese peptídica em fase sólida podem ser aplicadas na síntese de péptidos sob irradiação por micro-ondas, com resultados que superam geralmente os resultados obtidos sem irradiação. Conseguiram-se assim obter praticamente todos os derivados pretendidos, sendo que as maiores dificuldades foram encontradas nos péptidos com as α,α-dialquilglicinas mais estereoquimicamente impedidas, tal como esperado. A estratégia de síntese das α,α-dialquilglicinas usada baseia-se num procedimento de reação de Ugi seguida de acidólise, previamente estabelecido pelo grupo de trabalho em que se insere este trabalho. Foram também sintetizadas, com bons rendimentos, as N-fluoreniloxicarbonil-α,α-dialquilglicinas necessárias para a síntese dos péptidos, uma vez que não estão disponíveis comercialmente.

Analogues of Peptaibolin, a peptaibol with antibiotic activity, incorporating α,α-dialkylglycines (Deg, Dpg, and Ac6c) at selected positions were synthesised by MW-SPPS and fully characterized. A control analogue incorporating L-alanine was also prepared. The native peptide and the analogues were studied by fluorescence spectroscopy for their membrane permeating activity. Small unilamellar vesicles (SUVs) of egg phosphatidylcholine/ cholesterol (70:30) containing an encapsulated fluorescence probe (6-carboxyfluorescein) were used as membrane models. The assays of carboxyfluorescein release from SUVs upon peptide addition showed that Peptaibolin-Dpg and Peptaibolin-Ac6c are the most active peptides. These results indicate that the structure of the α,α-dialkylglycines is crucial for the membrane permeating ability of these Peptaibolin analogues.

In this paper, four natural deep eutectic solvents (NADES) systems were prepared at specific molar ratios, La:Bet (2:1) (lactic acid/betaine), La:Hist (9:1) (lactic acid/histidine), Ma:Bet:H2O (1:2:3) (malic acid/betaine/water) and Ma:Bet:Pro:H2O (1:1:1:2) (malic acid/betaine/proline/water). Their physical and thermodynamic properties were studied, namely viscosity, electrical conductivity, and heat capacity. The viscosity and electrical conductivity were determined as a function of temperature and the correlation for the temperature dependence was obtained and discussed based on Arrhenius theory. The heat capacity for all eutectic systems was measured by differential scanning calorimetry (DSC) over a temperature range of 293.15-363.15 K. The ability of these NADES to reduce cellulose crystallinity was evaluated. Cellulose crystallinity after suspension in these NADES was studied by X-ray diffraction. Cellulose suspended in Ma/Bet/H2O (1:2:3) suffer the highest crystallinity reduction among the systems studied and was about of 20%.

Natural deep eutectic systems (NADES) are mostly composed of natural primary metabolites such as sugars, sugar alcohols, organic acids, amino acids and amines. These simple molecules have been identified in animals living in environments with extreme temperature amplitudes, being responsible for their survival at negative temperatures during winter. Herein, we report for the first time the use of NADES based on trehalose (Treh) and glycerol (Gly) in cryopreservation, as cryoprotective agents (CPA). The evaluation of the thermal behaviour of these eutectic systems, showed that NADES have a strong effect on the water crystallization/freezing and melting process, being able to reduce the number of ice crystals and hence ice crystal damage in cells, which is a crucial parameter for their survival, upon freezing. Using this NADES as CPA, it is possible to achieve similar or even better cellular performance when compared with the gold standard for cryopreservation dimethyl sulfoxide (DMSO). In this sense, this work relates the physical properties of the NADES with their biological performance in cryopreservation. Our comprehensive strategy results in the demonstration of NADES as a promising nontoxic green alternative to the conventional CPA's used in cryopreservation methods.

The membrane permeabilization ability of a model peptaibol and several mimetics incorporating unnatural α,α- dialkylglycines were studied by fluorescence spectroscopy. The model membranes (egg phosphatidylcholine/cholesterol, 70:30) contained a fluorescent probe encapsulated. By addition of peptides at different peptide to lipid ratios, it was possible to follow the release of the encapsulated probe, monitoring the increase in fluorescence intensity over time using fluorescence spectroscopy.