Fluorescent and Photosensitizing Conjugates of Cell-Penetrating Peptide TAT(47-57): Design, Microwave-Assisted Synthesis at 60 °C, and Properties

Abstract

Conjugates based on cell-penetrating peptides (CPPs) are scientifically relevant owing to their structural complexity; their ability to enter cells and deliver drugs, labels, antioxidants, bioactive compounds, or DNA fragments; and, consequently, their potential for application in research and biomedicine. In this study, carboxyamidated fluorescently labeled conjugates FAM-GG-TAT(47-57)-NH2 and FAM-PEG6-TAT(47-57)-NH2 and photosensitizer-labeled conjugate Chk-PEG6-TAT(47-57)-NH2 [where TAT(47-57) is the CPP, 5(6)-carboxyfluorescein is the (FAM) fluorophore, chlorin k (Chk) is the photosensitizer, and the dipeptide glycyl–glycine (GG) or hexaethylene glycol (PEG6) is the spacer] were originally designed, prepared, and fully characterized. Practically, all chemical reactions of the synthetic steps (peptide synthesis, spacer incorporation, and conjugation) were microwave-assisted at 60 °C using optimized protocols to give satisfying yields and high-quality products. Detailed analyses of the conjugates using spectrofluorimetry and singlet oxygen detection showed that they display photophysical properties typical of FAM or Chk. Anticandidal activity assays showed that not only this basic property of TAT(47-57) was preserved in the conjugates but also that the minimal inhibitory concentration was slightly reduced for cells incubated with PS-bearing conjugate Chk-PEG6-TAT(47-57)-NH2. Overall, these results indicated that the synthetic approach on-resin assisted by microwaves at 60 °C is simple, straightforward, selective, metal-free, sufficiently fast, cleaner, and more cost-effective than those previously used for preparing this type of macromolecule. Furthermore, such new data show that microwaves at 60 °C and/or conjugation did not harm the integrity of the conjugates’ constituents. Therefore, FAM-GG-TAT(47-57)-NH2, FAM-PEG6-TAT(47-57)-NH2, and Chk-PEG6-TAT(47-57)-NH2 have high potential for practical applications in biochemistry, biophysics, and therapeutics.

Estudo da possibilidade de produção de dipeptídeos antimicrobianos por rota sintética verde

“…Hidrólise ácida total seguida de análise de aminoácidos do hidrolisado via HPLCQuantidades específicas do material a ser analisado foram submetidas à hidrólise gasosa ácida total a 130 °C em uma estação de trabalho Pico-Tag (Waters, USA), ambas na presença de uma solução 50 % (v/v) de HCl/ ácido propiônico por 24h sob atmosfera de N2.Os hidrolisados ácidos obtidos foram liofilizados, dissolvidos em água e filtrados para serem analisados em sistema de HPLC Dionex (Califórnia, EUA) composto por um amostrador automático (AS40), bomba quaternária (GS50), forno para a coluna (LC25), coluna de troca iônica (2 x 250 mm, AminoPac PA10), detector eletroquímico (ED50) e uma plataforma "Chromeleon" para controle e aquisição de dados. A identificação e quantificação foram feitas por análise comparativa de uma solução-padrão de 19 aminoácidos de concentrações conhecidas (DING; YU;MOU, 2002;OKUDA-SHINAGAWA et al, 2017). quantidade de reações testadas juntou-se as alíquotas pré-analisadasdos meios reacionais e o produto Z-Lys-Trp-OBzl foi fracionado por RP-HPLC (Figura 11).…”