CC BY
20Т 63 (5)
ИЗВЕСТИЯ ВЫСШИХ УЧЕБНЫХ ЗАВЕДЕНИЙ. Серия «ХИМИЯ И ХИМИЧЕСКАЯ ТЕХНОЛОГИЯ»
2020
IZVESTIYA VYSSHIKH UCHEBNYKH ZAVEDENII V 63 (5) KHIMIYA KHIMICHESKAYA TEKHNOLOGIYA 2020
RUSSIAN JOURNAL OF CHEMISTRY AND CHEMICAL TECHNOLOGY
DOI: 10.6060/ivkkt.20206305.6101 УДК: [547.759.5+577.112.34]:543.42
СИНТЕЗ И ИССЛЕДОВАНИЕ СПЕКТРАЛЬНЫХ СВОЙСТВ КОНЪЮГАТОВ BODIPY
С АМИНОКИСЛОТАМИ
К.В. Ксенофонтова, А.А. Ксенофонтов, И.А. Ходов, Е.В. Румянцев
Ксения Витальевна Ксенофонтова*
Ивановский государственный химико-технологический университет, Шереметевский пр., 7, Иваново, Российская Федерация, 153000 E-mail: kvk@isuct.ru*
Александр Андреевич Ксенофонтов
Институт химии растворов им. Г.А. Крестова РАН, ул. Академическая, 1, Иваново, Российская Федерация, 153045
E-mail: ivalex.09@mail.ru Илья Анатольевич Ходов
Институт химии растворов им. Г.А. Крестова РАН, ул. Академическая, 1, Иваново, Российская Федерация, 153045
Институт физики, Казанский федеральный университет, ул. Кремлевская, 18, Казань, Российская Федерация, 420008
E-mail: ilya.khodov@gmail.com Евгений Владимирович Румянцев
Ивановский государственный химико-технологический университет, Шереметевский пр., 7, Иваново, Российская Федерация, 153000
Ивановский государственный политехнический университет, Шереметевский пр., 21, Иваново, Российская Федерация, 153000 E-mail: naturer@yandex.ru
В настоящей работе описан направленный одностадийный синтез двух новых конъюгатов типа аминокислота - борфторидный комплекс дипиррометена (BODIPY), в которых остатки гистидина и тирозина связаны с остовом флуорофора через аминогруппу. Синтезированные вещества были полностью охарактеризованы посредством гН и UB спектроскопии ЯМР-, ИК-спектроскопии, времяпролетной масс-спектрометрии с мат-рично-активированной лазерной десорбцией/ионизацией. Все полученные данные находятся в соответствии с предполагаемыми структурами. Исследуемые конъюгаты BODIPY с аминокислотами были изучены методами спектрофото- и спектрофлуориметрии. Были получены спектры поглощения и испускания веществ в смеси диметилсульфоксид (ДМСО) -буферный раствор (БР) (1:1). Кроме того, был рассчитан ряд фотофизических характеристик конъюгатов, а именно: относительный квантовый выход флуоресценции, молярный коэффициент экстинкции, относительное время жизни возбужденного состояния, константы скорости излучательной и безызлучательной дезактивации. Были получены 3D спектры флуоресценции веществ в смеси ДМСО - БР (1:1). Было проведено спектрофото- и спектрофлуориметрическое титрование флуорофоров. Результаты титрования позволили нам дополнительно подтвердить стехиометрический состав конъюгатов BODIPY с аминокислотами. Был проведен квантовохимический анализ структурных и спектральных
свойств веществ в основном состоянии. Результаты квантовохимических расчетов позволили понять природу взаимодействий BODIPY с аминокислотой в конъюгатах. Более того, было обнаружено, что синтезированные конъюгаты BODIPY с аминокислотами обладают высокой растворимостью в полярных растворителях. Таким образом, ряд уникальных свойств объектов настоящего исследования обеспечивает возможность их использования для визуализации как биоактивных макромолекул, так и биохимических процессов в живых клетках.
Ключевые слова: BODIPY, аминокислоты, конъюгация, маркеры, флуоресценция
SYNTHESIS AND STUDY OF SPECTRAL PROPERTIES OF AMINO ACIDS - BODIPY CONJUGATES
K.V. Ksenofontova, A.A. Ksenofontov, I.A. Khodov, E.V. Rumyantsev
Ksenia V. Ksenofontova*
Ivanovo State University of Chemistry and Technology, Sheremetevskiy ave., 7, Ivanovo, 153000, Russia
E-mail: kvk@isuct.ru*
Alexander A. Ksenofontov
G. A. Krestov Institute of Solution Chemistry of the RAS, Akademicheskaya st., 1, Ivanovo, 153045, Russia
E-mail: ivalex.09@mail.ru
Ilya A. Khodov
G. A. Krestov Institute of Solution Chemistry of the RAS, Akademicheskaya st., 1, Ivanovo, 153045, Russia
Institute of Physics, Kazan Federal University, Kremlyovskaya st., 18, Kazan, 420008, Russia
E-mail: ilya.khodov@gmail.com
Evgeniy V. Rumyantsev
Ivanovo State University of Chemistry and Technology, Sheremetevskiy ave., 7, Ivanovo, 153000, Russia
Ivanovo State Politechnical University, Sheremetevskiy ave., 21, Ivanovo, 153000, Russia
E-mail: naturer@yandex.ru
The present work describes direct one-pot synthesis of the two novel amino acids - boron-dipyrromethene (BODIPY) conjugates with histidine and tyrosine residues bound to fluorophore via amino group. The synthesized compounds were fully characterized by means of 1H and 11B nuclear magnetic resonance spectroscopy, infrared spectroscopy, time-of-flight mass spectrome-try with matrix-activated laser desorption / ionization. All the data are in accordance with the proposed structures. The amino acids - BODIPY conjugates under study were investigated by means of absorbance and fluorescence spectroscopy. Absorption and emission spectra of the compounds in dimethyl sulfoxide (DMSO) - buffer solution (BS) mixture (1:1) were obtained. Moreover, a number of photophysical characteristics of the conjugates, namely: relative fluorescence quantum yield, molar extinction coefficient, relative fluorescence lifetime, radiative and non-radiative rate constants were calulated. 3D fluorescence spectra of the compounds in DMSO - BS mixture (1:1) were obtained. Absorption and emission titration of the compounds was carried out. The results of the titration allowed us to confirm additionally a stoichiometric composition of the conjugates. Quantum chemical analysis of structural and spectral properties of the compounds in a ground state was carried out. The results of quantum chemical calculations gave an insight to a nature of the BODIPY - amino acid interactions in the conjugates. Besides, the synthesized amino acids - BODIPY conjugates are found to be highly soluble in polar solvents. Thus, a range of unique properties of the objects of the present research enables us to use them for visualization of bioactive macromolecules as well as biochemical processes in living cells.
Key words: BODIPY, amino acids, conjugation, markers, fluorescence
Для цитирования:
Ксенофонтова К.В., Ксенофонтов А.А., Ходов И.А., Румянцев Е.В. Синтез и исследование спектральных свойств конъюгатов BODIPY с аминокислотами. Изв. вузов. Химия и хим. технология. 2020. Т. 63. Вып. 5. С. 4-11 For citation:
Ksenofontova K.V., Ksenofontov A.A., Khodov I.A., Rumyantsev E.V. Synthesis and study of spectral properties of amino acids - BODIPY conjugates. Izv. Vyssh. Uchebn. Zaved. Khim. Khim. Tekhnol. [Russ. J. Chem. & Chem. Tech.]. 2020. V. 63. N 5. P. 4-11
INTRODUCTION
One of the encouraging areas of modern science is creating fluorescent markers for compounds of biological origin. These objects, labeled with fluorescent molecules, find their application in a huge amount of life science researches [1-3]. In recent years, a class of boron-dipyrromethene (BODIPY) dyes has drawn scientific community attention due to the range of its special features: excellent quantum yield, high molar extinction coefficient, sharp absorption and emission peaks, and great ability to structural modification [4, 5]. All of these cause extensive use of BODIPY derivatives in biomolecule labeling [6].
Synthesis of conjugates based on BODIPY and some biomolecules is relatively new promising direction in chemistry. In this regard, one of the most interesting objects of research are amino acids -BODIPY conjugates. This type of bioconjugates can be applied for visualization of various biochemical processes, such as protein interactions with other bi-omolecules, protein localization and dynamics, enzyme activity, etc. [7-9].
There is currently not a great number of scientific papers devoted to a development of amino acids - BODIPY conjugates. The only works on this subject are [10-12]. Guzow et al. [10] synthesized an alanine-BODIPY conjugate which can be utilized in peptide synthesis. Mendive-Tapia et al. [11] obtained a tryptophan-BODIPY conjugate and incorporated it into several peptides for ex vivo imaging of human tissues. Our scientific group [12] earlier synthesized lysine-, methionine-, and tryptophan-BODIPY conjugates which can be used in bioimaging.
It is noteworthy that conjugates based on BODIPY and other classes of small biomolecules are extensively studied. For example, there are bioconjugates of BODIPY derivatives and nucleotides [13, 14], lipids [15-17], hormones [18, 19], vitamins [20], etc.
The present work describes synthesis and characterization of two novel amino acids - BODIPY conjugates with histidine and tyrosine residues bound to fluorophore via amino group. Spectral properties of the obtained compounds were investigated. Advantageous photophysical characteristics and high solubility in polar solvents make these conjugates promising fluorescent markers for various biological objects.
EXPERIMENTAL
Materials
The reagents and solvents were obtained from commercial sources (Sigma-Aldrich, Fisher Chemical, Lab-Scan, Reakhim, Khimmed) and used without further purification.
Instruments
1H and 11B nuclear magnetic resonance (NMR) spectra were obtained by means of a Bruker Avance III 500 NMR spectrometer. Infrared (IR) spectra were recorded on a Bruker VERTEX 80v FTIR spectrometer. Matrix-assisted laser desorp-tion/ionization (MALDI) time of flight (TOF) mass spectra were registered by a Shimadzu AXIMA Confidence MALDI TOF-TOF mass spectrometer. Ab-sorbance and fluorescence spectroscopy experiments were performed on a Solar SM 2203 spectrofluorome-ter and an Agilent Cary Eclipse fluorescence spectro-photometer.
Scheme 1. Scheme of amino acids - BODIPY conjugates synthesis Схема 1. Схема синтеза конъюгатов BODIPY с аминокислотами
Synthesis of amino acids - BODIPY conjugates
The succinimidyl ester of 3-(4,4-difluoro-2,6-dimethyl -4 -bora-3a,4a-diaza-s -indacene-8-yl)-pro -pionic acid (NHS-BODIPY) (1 mg, 2.57 ^mol, 1 equiv.) synthesized following the procedure [12] was dissolved in 10 ml of dimethyl sulfoxide (DMSO). An amino acid (2.57 ^mol, 1 equiv.) was dissolved in 10 ml of NaHCO3 - NaOH buffer solution (BS) [21] with pH of 11.0. At this pH, more than 90% of amino acid species in aqueous solutions are anionic ones, which are perfect for binding to the amine-reactive NHS-BODIPY. The mixture of the NHS-BODIPY in DMSO and an amino acid in BS was stirred in an ice bath for 12 h. After the end of the reaction, the mixture was washed with dichloromethane (DCM) to eliminate the unreacted NHS-BODIPY. The solvents were removed under reduced pressure to give the pure products HisN-BODIPY and TyrN-BODIPY (Scheme 1).
N6-(3-(4,4-Difluoro-2,6-dimethyl-4-bora-3a,4a-diaza-s-indacene-8-yl)propanoyl)-L-hystidine HisN-BODIPY: Light brown solid (1.1 mg, 97%). :H NMR (500.17 MHz, D2O - H2O mixture (1:9)): S (ppm) 8.97 (dd, 1H, J = 7.6, 6.6 Hz, >NHammo acid), 8.45 (dd, 1H, J = 7.6, 2.1 Hz, -CH=amlno acid), 8.30 (s, 1H, -NH-amide), 7.34-7.31 (m, 1H, CH=amino acid), 4.47 (dt, 1H, J = 13.0, 7.0 Hz, >CH-^ acid), 3.82 (td, 2H, J = 7.1, 1.6 Hz, -CH2-), 3.47 (t, 2H, J = 7.1 Hz, -CH2-), 3.10-2.96 (m, 2H, -CH-amno acid), 2.58 (d, 3H, J = 1.0 Hz, -CH3), 2.39 (t, 3H, J = 0.6 Hz, -CH3). nB NMR (160.48 MHz, D2O - H2O mixture (1:9)): S (ppm) 2.3 (br, 1B, -BF2-). IR (KBr): v (cm-1) 3414 (w, br, N-Hamide), 2974-2794 (w, br, C-H), 1690 (w, C=Oamide), 1621 (w, C-Namide, N-Hamide), 1440 (s, sh, C-Namide), 1057 (w, B-F). MS (MALDI TOF, a-cyano-4-hydroxycinnamic acid (CHCA) matrix): m/z calculated for C20H21BF2KN5O3 [M-H+K] 467.13, found 466.89.
N-(3-(4,4-Difluoro-2,6-dimethyl-4-bora-3a,4a-diaza-s-indacene-8-yl)propanoyl)-L-tyrosine TyrN-BODIPY: Light brown solid (1.2 mg, 99%). 1H NMR (500.17 MHz, D2O - H2O mixture (1:9)): S (ppm) 8.31 (s, 1H, J = 0.9 Hz, -NH-amide), 7.84 (s, 1H, -OHamino acid), 7.05-6.99 (m, 2H, -CH amino acid), 6.71-6.64 (m, 2H, CH=amino acid), 4.42 (dt, 1H, J = 12.0, 7.0 Hz, >CH-amino acid), 3.86 (td, 2H, J = 7.1, 2.1 Hz, -CH2-), 3.53-3.38 (m, 2H, -CH2-), 3.09-2.97 (m, 2H, CH2 amino acid), 2.58 (t, 3H, J = 1.0 Hz, -CH3), 2.31 (s, 3H, -CH3). 11B NMR (160.48 MHz, D2O -H2O mixture (1:9)): S (ppm) 2.7 (br, 1B, -BF2-). IR (KBr): v (cm-1) 3470 (m, br, N-Hamide), 2956-2808 (w, br, C-H), 1692 (m, C=Oamide), 1624 (w, C-Namide, N-Hamide), 1461 (s, sh, C-Namide), 1060 (w, B-F), 652 (m, N-Hamide), 610 (m, N-Hamide). MS (MALDI TOF, 2,5-dihydroxybenzoic acid (DHB) matrix): m/z calculated for C23H22BF2Li2N3O4 [M-2H+2Li] 467.20, found 466.91.
Computational details
The quantum chemical calculations of the investigated molecules were carried out on the basis of the methodology described in [12].
Determination of photophysical characteristics
The fluorescence quantum yields O of the fluorophores under study were estimated by comparison with Rhodamine 6G as a standard of the known quantum yield = 94 % in ethanol [22]) using the equation (1) [23].
A —7.
(1)
ф = ф jk^klk ^x ^st - •
Sst Ax n|t
where 0 is the fluorescence quantum yield, S is the integrated area under the emission spectrum, A is the absorbance at the excitation wavelength, n is the refractive index of the solvent. The subscripts x and st refer to unknown and reference solutions, respectively.
The actual fluorescence lifetimes t were computed by the PhotochemCAD program [24-26] which is based on the Strickler - Berg approach [27].
The radiative kr and non-radiative knr rate constants were calculated using the equations (2) and (3) [23], respectively.
~ (2)
(3)
— ' T
к — лnr
1-ф
ids
RESULTS AND DISCUSSION
Absorption and emission spectra of amino ac-BODIPY conjugates
It was obtained absorption and emission spectra along with a number of photophysical characteristics of the amino acids - BODIPY conjugates HisN-BODIPY and TyrN-BODIPY in DMSO - BS mixture (1:1) (Fig. 1, Table).
1,0-i
0,8-
0,6-
0,4-
0,2-
0,0
1,0
0,8
0,6
0,4
0,2
0,0
300
400 500
Я, nm
600
700
Fig. 1. Normalized absorption and emission spectra of NHS-BODIPY [12] (1 and 1', respectively), HisN-BODIPY (2 and 2', respectively), and TyrN-BODIPY (3 and 3', respectively) in DMSO - BS mixture (1:1) Рис. 1. Нормализованные спектры поглощения и флуоресценции NHS-BODIPY [12] (1 и 1', соответственно), HisN-BODIPY (2 и 2', соответственно) и Tyi^-BODIPY (3 и 3', соответственно) в смеси диметилсульфоксид (ДМСО) - буферный раствор (БР) (1:1)
Table
Photophysical characteristics of NHS-BODIPY [12], HisN-BODIPY, and TyrN-BODIPY in DMSO - BS mixture (1:1)
Таблица. Фотофизические характеристики NHS-BODIPY [12], HisN-BODIPY и TyrN-BODIPY в смеси ДМСО - БР (1:1)
NHS-BODIPY [12] HisN-BODIPY Tyr -BODIPY
ф, % 92 33 29
е, l/mol cm 64700 7100 3400
т, ns 3.88 3.71 10.60
kr, ns-1 0.237 0.089 0.027
knr, ns 0.021 0.181 0.066
The absorption spectra of the amino acids -BODIPY conjugates give three absorption bands: 291, 426, and 506 nm for the HisN-BODIPY and 282, 450, and 508 nm for the TyrN-BODIPY. The emission spectra give one band with the maximum absorption wavelength of 518 nm in the case of the HisN-BODIPY and two bands with the maxima absorption wavelengths of 306 and 519 nm in the case of the TyrN-BODIPY. The HisN-BODIPY and the TyrN-BODIPY exhibit relatively small Stokes shifts of 12 and 11 nm, respectively. The BODIPY characteristic absorption and emission bands maxima of the conjugates are 2-4 nm bathochromically shifted compared with the spectra of the NHS-BODIPY.
It is observed a significant three times decrease of the fluorescence quantum yields of the HisN-BODIPY and the TyrN-BODIPY in contrast to that of the NHS-BODIPY. This fact can be explained by several reasons [23]. Firstly, the decrease of the fluorescence quantum yields of the conjugates is connected with an increase of the molecular size resulting in an increase of an internal conversions probability. Secondly, this is due to the photoinduced electron transfer (PeT) process occurring (see section Quantum chemical calculations of BODIPY precursor and amino acids - BODIPY conjugates).
During the work, it was discovered that the amino acids - BODIPY conjugates have high solubility in a number of polar solvents, including water. Unfortunately, spectral properties of the bioconju-gates in water have not been investigated within the scope of the present study but this research is the aim of our further work.
3D Fluorescence spectra of BODIPY precursor and amino acids - BODIPY conjugates
It was obtained 3D fluorescence spectra of the BODIPY precursor NHS-BODIPY and the amino acids - BODIPY conjugates HisN-BODIPY and TyrN-BODIPY in DMSO - BS mixture (1:1) (Fig. 2).
Fig. 2. 3D Fluorescence spectra of NHS-BODIPY (a), HisN-BODIPY
(b), and TyrN-BODIPY (c) in DMSO - BS mixture (1:1) Рис. 2. 3D Спектры флуоресценции NHS-BODIPY (a), HisN-BODIPY (b) и TyrN-BODIPY (c) в смеси ДМСО - БР (1:1)
c
3D Fluorescence spectra, obtained by measuring fluorescence emission via scanning both emission and excitation wavelengths, are a valuable tool for analyzing fluorophores. These spectra are consider to be much more useful to discriminate among similar fluorescent dyes than individual numerical data [28].
The 3D fluorescence spectrum of the NHS-BODIPY gives two characteristic excitation / emission peaks: Xex/Xem = 350/515 and 500/515 nm. The fluorescence intensity is about ten times higher at Xx = 500 nm than 350 nm. The peaks mentioned above are due to S0—S2 and S0-S1 electron transitions, respectively.
The 3D fluorescence spectrum of the HisN-BODIPY gives two characteristic excitation / emission peaks: Xex/Xem = 280/500 and 488/500 nm. The fluorescence intensity is about three times higher at Xx = 488 nm than 280 nm. The peaks mentioned above are due to S0-S2 and S0-S1 electron transitions, respectively. The 3D fluorescence spectrum of the TyrN-BODIPY gives three characteristic excitation / emission peaks: two peaks with XJXem = 280/519 and 500/519 nm and one new peak with Xex/Xem = 280/306 nm. The fluorescence intensity at 519 nm is about five times higher at Xx = 500 nm than 280 nm. The first two peaks are due to S0-S2 and S0-Si electron transitions, respectively, while the third peak is due to S0-S3 electron transition (see section Quantum chemical calculations of BODIPY precursor and amino acids -BODIPY conjugates) and is connected to tyrosine residue fluorescence [29].
Thus, the results of 3D fluorescence spectroscopy are in good agreement with the results of ab-sorbance and 2D fluorescence spectroscopy as well as quantum chemical calculations (see sections Absorption and emission spectra of amino acids - BODIPY conjugates and Quantum chemical calculations of BODIPY precursor and amino acids - BODIPY conjugates).
Absorption and emission titration of amino acids - BODIPY conjugates
It was carried out absorbance and fluorescence titration of the NHS-BODIPY dissolved in DMSO by an amino acid dissolved in BS. Fig. 3 shows the results of fluorescence titration of the NHS-BODIPY by the His as an example. The trends mentioned below are similar for the Tyr and the other types of titration and that is why are not described here.
During the fluorescence titration, it is observed a weak 1 nm bathochromic shift of the emission bands maxima with increasing His concentration. An initial addition of the amino acid causes a strong fluorescence quenching due to an orientation interaction between the BODIPY and the His [12]. At equal
concentration of the components, there is the inflection point on the titration curve (Fig. 3(6)) that indicates the formation of the His-BODIPY conjugate of stoichiometric composition.
500
400
300
200-
100-
500
550 600
A, nm
1,6
1,4-
1,2
1,0
0,8
1:1
0
1
2
3
4
5
6
7
[His] / [NHS-BODIPY] b
Fig. 3. Emission spectra of NHS-BODIPY in DMSO with increasing His concentration in BS ([His] / [NHS-BODIPY]: 0 - 0.0; 1 - 0.2; 2 - 0.4; 3 - 0.6; 4 - 1.0; 5 - 2.0; 6 - 3.0; 7 - 4.0; 8 - 5.0; 9 - 6.0; 10 - 7.0) (a) and dependence of a ratio ofNHS-BODIPY fluorescence intensity at maximum emission wavelength without (I0) and with (Ii) an addition of His on a molar ratio of components ([His] / [NHS-BODIPY]) (b)
Рис. 3. Спектры флуоресценции NHS-BODIPY в ДМСО при увеличении концентрации His в БР ([His] / [NHS-BODIPY]: 0 - 0,0; 1 - 0,2; 2 - 0,4; 3 - 0,6; 4 - 1,0; 5 - 2,0; 6 - 3,0; 7 - 4,0; 8 - 5,0; 9 - 6,0; 10 - 7,0) (a) и зависимость отношения интенсивности флуоресценции NHS-BODIPY на длине волны максимума испускания без (I0) и с (Ii) добавлением His от молярного соотношения компонентов ([His] / [NHS-BODIPY]) (b)
0
a
Quantum chemical calculations of BODIPY precursor and amino acids - BODIPY conjugates
It was carried out the quantum chemical analysis of structural and spectral properties of the BOD-IPY precursor NHS-BODIPY and the amino acids -BODIPY conjugates HisN-BODIPY and TyrN-BODIPY in a ground state for interpreting a nature of spectral properties of the fluorophores.
TDDFT analysis results are presented in Fig. 4.
Fig. 4. Frontier molecular orbitals (FMOs) distributions of NHS-BODIPY (a), HisN-BODIPY (b), and TyrN-BODIPY (c) (B3LYP-D3(PCM)/def2-TZVP) Рис. 4. Распределение граничных молекулярных орбиталей NHS-BODIPY (a), HisN-BODIPY (b) и TyrN-BODIPY (c) (B3LYP-D3(PCM)/def2-TZVP)
There are three bands of different intensities in the TDDFT spectra of the amino acids - BODIPY conjugates HisN-BODIPY and TyrN-BODIPY, as opposed to the BODIPY precursor NHS-BODIPY with two bands. The experimental results exhibit exactly the same trends (see section Absorption and emission
spectra of amino acids - BODIPY conjugates). All the spectral bands are due to S0-Sn electron transitions.
In the case of the NHS-BODIPY, S0-S1 band has a maximum at 413 nm, while S0-S2 band - at 305 nm. Both transitions are caused by intramolecular n-n* energy transfer in a BODIPY moiety.
In the case of the HisN-BODIPY and the TyrN-BODIPY, S0-S1 band has a maximum in the range of 413-416 nm, S0-S2 band - in the range of 307-310 nm, and S0-S3 band - at 279 nm. The dominating contribution to spectral properties of the conjugates is made by the highest occupied molecular orbital (HOMO) - lowest unoccupied molecular orbital (LUMO) transition with the oscillator strength of 61-62%. Both S0-Si and S0-S2 transitions are caused by intramolecular n-n* energy transfer in a BODIPY moiety of a conjugate. HOMO-5 - LUMO transition in the HisN-BODIPY molecule and HOMO-6 - LU-MO transition in the TyrN-BODIPY molecule are caused by PeT from an electron donating amino acid moiety of a conjugate to an electron withdrawing BOD-IPY one (PeT mechanism).
CONCLUSIONS
The two novel fluorescent amino acids -BODIPY conjugates HisN-BODIPY and TyrN-BODIPY were obtained through a direct one-step synthesis and thoroughly characterized using 1H and 11B NMR-spectroscopy, IR-spectroscopy, MALDI-TOF-spectrometry. Spectral properties of the amino acids - BODIPY conjugates HisN-BODIPY and TyrN-BODIPY were investigated by absorbance spectros-copy, 2D and 3D fluorescence spectroscopy, computer modeling. The synthesized amino acids - BODIPY conjugates HisN-BODIPY and TyrN-BODIPY are found to be highly soluble in polar solvents. This valuable feature allows us to expect an increase in their bioavailability and affinity for peptides and proteins and hence their potential use as fluorescent markers for various biological objects.
ACKNOWLEDGMENTS
This research was partially supported by Russian Foundation for Basic Research (projects numbers: 18-33-20218 mol_a_ved, 18-43-370035 r_a, 1829-06008 mk). The authors gratefully acknowledge use of the services and facilities of the Centre for joint use of scientific equipment of Ivanovo State University of Chemistry and Technology and the Centre for joint use of scientific equipment "The upper Volga region centre of physico-chemical research". The authors are grateful to Joint Supercomputer Center of the Russian Academy of Sciences for providing MVS-100K supercomputer resources.
c
REFERENCES ЛИТЕРАТУРА
1. Gon^alves M.S.T. Fluorescent labeling of biomolecules with organic probes. Chem. Rev. 2009. V. 109. N 1. P. 190212. DOI: 10.1021/cr0783840.
2. Vahrmeijer A.L., Hutteman M., van der Vorst J.R., van de Velde C.J.H., Frangioni J.V. Image-guided cancer surgery using near-infrared fluorescence. Nat. Rev. Clin. Oncol. 2013. V. 10. N 9. P. 507-518. DOI: 10.1038/nrclinonc.2013.123.
3. Schneckenburger H., Reuter B.W., Schoberth S.M. Fluorescence techniques in biotechnology. Trends Biotechnol. 1985. V. 3. N 10. P. 257-261. DOI: 10.1016/0167-7799(85)90025-3.
4. Wood T.E., Thompson A. Advances in the chemistry of dipyrrins and their complexes. Chem. Rev. 2007. V. 107. N 5. P. 1831-1861. DOI: 10.1021/cr050052c.
5. Loudet A., Burgess K. BODIPY dyes and their derivatives: Syntheses and spectroscopic properties. Chem. Rev. 2007. V. 107. N 11. P. 4891-4932. DOI: 10.1021/cr078381n.
6. Kowada T., Maeda H., Kikuchi K. BODIPY-based probes for the fluorescence imaging of biomolecules in living cells. Chem. Soc. Rev. 2015. V. 44. N 14. P. 4953-4972. DOI: 10.1039/C5CS00030K.
7. Lavis L.D., Raines R.T. Bright ideas for chemical biology. ACS Chem. Biol. 2008. V. 3. N 3. P. 142-155. DOI: 10.1021/cb700248m.
8. Krueger A.T., Imperiali B. Fluorescent amino acids: Modular building blocks for the assembly of new tools for chemical biology. Chem.BioChem. 2013. V. 14. N 7. P. 788799. DOI: 10.1002/cbic.201300079.
9. Marfin Yu.S., Solomonov A.V., Timin A.S., Rumyantsev E.V. Recent advances of individual BODIPY and BODIPY-based functional materials in medical diagnostics and treatment. Curr. Med. Chem. 2017. V. 24. N 25. P. 2745-2772. DOI: 10.2174/0929867324666170601092327.
10. Guzow K., Kornowska K., Wiczk W. Synthesis and photo-physical properties of a new amino acid possessing a BOD-IPY moiety. Tetrahed. Lett. 2009. V. 50. N 24. P. 29082910. DOI: 10.1016/j.tetlet.2009.03.195.
11. Mendive-Tapia L., Zhao C., Akram A.R., Preciado S., Albericio F., Lee M., Serrels A., Kielland N., Read N.D., Lavilla R., Vendrell M. Spacer-free BODIPY fluorogens in antimicrobial peptides for direct imaging of fungal infection in human tissue. Nat. Commun. 2016. V. 7. Article number: 10940. DOI: 10.1038/ncomms10940.
12. Ksenofontova K.V., Ksenofontov A.A., Khodov I.A., Rumyantsev E.V. Novel BODIPY-conjugated amino acids: Synthesis and spectral properties. J. Mol. Liq. 2019. V. 283. P. 695-703. DOI: 10.1016/j.molliq.2019.03.148.
13. Dziuba D., Jurkiewicz P., Cebecauer M., Hof M., Hocek M. A rotational BODIPY nucleotide: An environmentsensitive fluorescence-lifetime probe for DNA interactions and applications in live-cell microscopy. Angew. Chemie Int. Ed. 2016. V. 55. N 1. P. 174-178. DOI: 10.1002/anie.201507922.
14. Seo T. S., Bai X., Kim D. H., Meng Q., Shi Sh., Ruparel H., Li Z., Turro N. J., Ju J. Four-color DNA sequencing by synthesis on a chip using photocleavable fluorescent nucleo-
tides. Proc. Natl. Acad. Sci. 2005. V. 102. N 17. P. 59265931. DOI: 10.1073/pnas.0501965102.
15. Boldyrev I.A., Zhai X., Momsen M.M., Brockman H.L., Brown R.E., Molotkovsky J.G. New BODIPY lipid probes for fluorescence studies of membranes. J. Lipid Res. 2007. V. 48. N 7. P. 1518-1532. DOI: 10.1194/jlr.M600459-JLR200.
16. Li Z., Mintzer E., Bittman R. First synthesis of free cho-lesterol-BODIPY conjugates. J. Org. Chem. 2006. V. 71. N 4. P. 1718-1721. DOI: 10.1021/jo052029x.
17. Marks D.L., Bittman R., Pagano R.E. Use of Bodipy-labeled sphingolipid and cholesterol analogs to examine membrane microdomains in cells. Histochem. Cell Biol. 2008. V. 130. N 5. P. 819-832. DOI: 10.1007/s00418-008-0509-5.
18. Osati S., Ali H., van Lier J.E. BODIPY-steroid conjugates: Syntheses and biological applications. J. Porphyr. Phthal. 2016. V. 20. N 01n04. P. 61-75. DOI: 10.1142/S1088424616300019.
19. Malachowska-Ugarte M., Sperduto C., Ermolovich Yu.V., Sauchuk A.L., Jurasek M., Litvinovskaya R.P., Straltsova D., Smolich I., Zhabinskii V.N., Drasar P., Demidchik V., Khripach V.A. Brassinosteroid-BODIPY conjugates: Design, synthesis, and properties. Steroids. 2015. V. 102. P. 53-59. DOI: 10.1016/j.steroids.2015.07.002.
20. West R., Panagabko C., Atkinson J. Synthesis and characterization of BODIPY-a-tocopherol: A fluorescent form of vitamin E. J. Org. Chem. 2010. V. 75. N 9. P. 2883-2892. DOI: 10.1021/jo100095n.
21. Rumble J.R. CRC Handbook of Chemistry and Physics. CRC Press, Taylor & Francis Group, an Informa Group company. 2017. 2560 p.
22. Fischer M., Georges J. Fluorescence quantum yield of rhodamine 6G in ethanol as a function of concentration using thermal lens spectrometry. Chem. Phys. Lett. 1996. V. 260. N 1-2. P. 115118. DOI: 10.1016/0009-2614(96)00838-X.
23. Lakowicz J.R. Principles of Fluorescence Spectroscopy. Springer Science+Business Media, LLC. 2006. 954 p.
24. Du H. Fuh R.-Ch.A., Li J., Corkan L.A., Lindsey J.S. Photo-chemCAD: A computer-aided design and research tool in photochemistry. Photochem. Photobiol. 1998. V. 68. N 2. P. 141142. DOI: 10.1111/j.1751-1097.1998.tb02480.x.
25. Dixon J.M., Taniguchi M., Lindsey J.S. PhotochemCAD 2: A refined program with accompanying spectral databases for photochemical calculations. Photochem. Photobiol. 2007. V. 81. N
1. P. 212-213. DOI: 10.1111/j.1751-1097.2005.tb01544.x.
26. Taniguchi M., Du H., Lindsey J.S. PhotochemCAD 3: Diverse modules for photophysical calculations with multiple spectral databases. Photochem. Photobiol. 2018. V. 94. N
2. P. 277-289. DOI: 10.1111/php.12862.
27. Strickler S.J., Berg R.A. Relationship between absorption intensity and fluorescence lifetime of molecules. J. Chem. Phys. 1962. V. 37. N 4. P. 814-822. DOI: 10.1063/1.1733166.
28. Soltzberg L.J., Lor S., Okey-Igwe Nn., Newman R. 3D Fluorescence characterization of synthetic organic dyes. Am. J. Anal. Chem. 2012. V. 3. N 9. P. 622-631. DOI: 10.4236/ajac.2012.39081.
29. Grigoryan K.R., Shilajyan H.A. Fluorescence 2D and 3D spectra analysis of tryptophan, tyrosine and phenylalanine. Proc. Yerevan State Univ. 2017. V. 51. N 1. P. 3-7.
Поступила в редакцию (Received) 02.07.2019 Принята к опубликованию (Accepted) 12.03.2020
