Spectral-luminescent properties of products of interaction of polyfluorinated containing a pyrazoline fragment pyrylium dyes with bovine serum albumin and amino acids

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Abstract

Studies of reaction ability fluorescent dyes for the production of labeled proteins and amino acids are important for the fields of bioengineering and biomedicine, in particular for obtaining bioimages for the purpose of cell visualization, studying the structure of labeled proteins by biophysical methods. Pyrylium dyes are able to interact with amino groups of proteins to form luminescent products, which allows them to be used in the field of proteomics. It is interesting to study the conjugation pyrylium dyes, containing both polyfluorinated fragment responsible to increased lipophilicity at proteins conjugation and pyrazoline fragment demonstrating anticancer activity. Pyrylium dyes containing a pyrazoline fragment and dialkylamino substituents (piperidino-, dibutylamino-, 4-hydroxypiperidino-) in a polyfluorinated aromatic ring in the donor part were synthesized by Knoevenagel condensation reaction. The reaction of pyrylium dyes with compounds containing a primary amino group was carried out to obtain a pyridinium dyes by the ANRORC mechanism (Addition of Nucleophiles, Ring Opening and Ring Closure). The ability of pyrуlium dyes to react with bovine serum albumin (BSA) and amino acids such as Lys, Arg, Cys, Phe to form pyridinium luminophore was shown. The spectral-luminescent properties of the resulting luminophores were investigated. The product of the reaction of pyrуlium dye (Е)-2,6-dimethyl-4-(4-{3-phenyl-5-[2,3,5,6-tetrafluorо-4-(piperidine-1-yl)phenyl]-4,5-dihydro-1Н-pуrazole-1-yl}-styryl)pyrylium tetrafluoroborate with Lys was isolated and its structure was confirmed by NMR spectroscopy. The binding site of pyrylium dyes with BSA – e-amino group of Lys was determined. Along with pyridinium luminophores, in aqueous solutions hydrolysis products are formed that are not bonded with protein and absorb in the short-wavelength region. The calculated amount of luminophore bound to BSA is two molecules of pyrylium dye per one molecule of BSA. The synthesized pyrylium dyes react with BSA in the mixture of phosphate buffer with methanol (pH 7.4) 3–4 orders of magnitude faster than the well-known julolidine dye Py-1. The relative reaction rates of (Е)-2,6-dimethyl-4-(4-{3-phenyl-5-[2,3,5,6-tetrafluorо-4-(4-hydroxypiperidine-1-yl)phenyl]-4,5-dihydro-1Н-pуrazole-1-yl}styryl) pyrуlium tetrafluoroborate with amino acids were determined as Lys > Cys >> Phe ≥ Arg. The obtained polyfluoro pyrylium-pyrazolinium dyes have the application perspective in the field of bioimaging, proteomic and biomedicine, due to high conjunction rate and efficiency with BSA and amino acids.

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About the authors

V. V. Shelkovnikov

Novosibirsk Institute of Organic Chemistry, Siberian Branch, Russian Academy of Sciences; Novosibirsk State Technical University

Author for correspondence.
Email: vice@nioch.nsc.ru
Russian Federation, Novosibirsk; Novosibirsk

D. D. Doroshenko

Novosibirsk Institute of Organic Chemistry, Siberian Branch, Russian Academy of Sciences

Email: vice@nioch.nsc.ru
Russian Federation, Novosibirsk

I. Yu. Kargapolova

Novosibirsk Institute of Organic Chemistry, Siberian Branch, Russian Academy of Sciences

Email: vice@nioch.nsc.ru
Russian Federation, Novosibirsk

P. A. Kozlakov

Novosibirsk State Technical University

Email: vice@nioch.nsc.ru
Russian Federation, Novosibirsk

N. A. Shestakov

Novosibirsk State Technical University

Email: vice@nioch.nsc.ru
Russian Federation, Novosibirsk

Yu. S. Sotnikova

Novosibirsk Institute of Organic Chemistry, Siberian Branch, Russian Academy of Sciences

Email: vice@nioch.nsc.ru
Russian Federation, Novosibirsk

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Supplementary files

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1. JATS XML
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2. Fig. 1. Structures of fluorine-containing pyrilium-pyrazoline dyes and the yulolidine dye Py-1.

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3. Fig. 2. Normalized electronic absorption spectra of the PDC-Pip-Lys product (1) in a phosphate buffer/EtOH mixture (10:1) and the starting dye PLC-Pip in EtOH (2).

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4. Fig. 3. Excitation spectra of PDC-Pip-BSA (483 nm) and luminescence spectra of PDC-Pip-BSA (596 nm), PDC-Pip-Lys (598 nm), PDC-Pip-Arg (620 nm), PDC-Pip-Cys (613 nm), PDC-Pip-Phe (606 nm) in a phosphate buffer/EtOH (10:1) mixture.

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5. Fig. 4. Excitation (λmax=461 nm), emission spectra of PDC-Pip-OH-Lys (λmax=606 nm) and PDC-Pip-OH-Lys upon addition of an equimolar solution of BSA (λmax=576 nm), in a mixture of phosphate buffer/EtOH (10:1).

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6. Fig. 5. Electronic absorption spectrum of PDC-Pip-BSA in phosphate buffer/EtOH (10:1) mixture.

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7. Fig. 6. Electronic absorption spectrum of BSA (1) in a phosphate buffer/EtOH (10:1) mixture and the PLC-Pip hydrolysis product (2) in EtOH.

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8. Fig. 7. Evolution of electronic absorption spectra in a phosphate buffer/MeOH (10:1) mixture during the interaction of (a) PLC-Pip-OH with BSA with an interval between spectrum recordings of 5 s and (b) Py-1 with BSA with an interval between spectrum recordings of 1 h.

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9. Fig. 8. (a) – Evolution of electronic absorption spectra in the AcOH/MeOH/Et3N mixture during the interaction of PLC-Pip-OH with Lys with an interval between spectral recordings of 30 min; (b) – linear approximation by the pseudo-first-order equation of the experimental points of the kinetic curve of accumulation of the PDC-Pip-OH-Lys product, recorded with an interval of 5 min at the maximum of the absorption spectrum.

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10. Scheme 1. Reactions of compounds (Ia–c) with 2,4,6-trimethylpyrylium tetrafluoroborate in MeOH.

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11. Scheme 2. Reaction of PLC-Pip with Lys in MeOH.

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