CC BY
8УДК 541
.11/.118
Dmitriy S. Shevchenko1, Olga V. Rakhimova2, Nikolay A. Charykov3, Konstantin N. Semenov4, Victor A. Keskinov5, Alexander L. Vorobiev6, Natalie A. Kulenova7, Zhanar S. Onalbaeva8
SYNTHESIS, ID ENTIFICATION AND BIOTESTING OF WATER SOLUBLE OCTO-ADDUCT OF FULLERENE C60 AND ARGININE C60(C6H12NaN4O2)8H8
St. Petersburg Electrotechnical University «LETI», ul. Professora Popova 5, 197376, St. Petersburg, Russia St. Petersburg State Institute of Technology (Technical University), Moskovsky pr., 26 Saint-Petersburg, 190013, Russia St Petersburg State University,7/9 Universitetskaya emb., Saint-Petersburg, 199034, Russia
D. Serikbayev East Kazakhstan state technical university, A.K. Protozanov Street, 69, Ust-Kamenogorsk city, 070004, Republic of Kazakhstan. e-mail: keskinov@mail.ru
The synthesis, identification by the methods of element analysis, ffrared and electronic spectroscopy, complex thermal analysis of water soluble octo-adduct of light fullerene C60 with L-arginine C60(C6H12NaN402)8H8 are reported. Using as the test micro-organism chlorella vulgaris beijer, bio-testing ofthe adduct investigated.
Key words: fullerene C60, arginine, octo-adduct, synthesis, identification, bio-testing, chlorella vulgaris beijer
Introduction
Light fullerenes (C60 and C70) may be more or less effectively used in different fields of science and technics,
Д.С. Шевченко1 , О.В. Рахимова2 , Н.А. Чарыков3 , К.Н. Семенов4 , В.А. Кескинов5 , А.Л. Воробьев 6, Н.А. Куленова7 , Ж.С. Оналбаева8
СИНТЕЗ, ИДЕНТИФИКАЦИЯ И БИОТЕСТИРОВАНИЕ ВОДОРАСТВОРИМОГО ОКТО-АДДУКТА ФУЛЛЕРЕНА C60 И
АРГИНИНА 60
C60(C6H12NAN4O2)8H8
Санкт-Петербургский государственный электротехнический университет «ЛЭТИ», ул. Профессора Попова, 5, Санкт-Петербург, 197376, Санкт-Петербург, Россия Санкт-Петербургский государственный технологический институт (технический университет), Московский пр. 26, Санкт-Петербург, 190013, Россия
Санкт-Петербургский государственный университет, Университетская наб., 7/9, Санкт-Петербург, 199034, Россия Восточно-Казахстанский государственный технический университет им. Д Серикбаева, ул. Протозанова, 69, г. Усть-Каменогорск, 070004, Республика Казахстан e-mail: keskinov@mail.ru
Сообщается о синтезе, идентификации методами элементного анализа, инфракрасной и электронной спектроскопии, комплексного термического анализа водорастворимого окто-аддукта легкого фуллерена C60 с L-аргинином C60(C6H12NaN402)8H8. Био тестирование исследуемого аддукта проводилось с использованием в качестве тестового микроорганизма chlorella vulgaris Ьецег(хлорелла обыкновенная).
Ключевые слова: фуллерен С60, аргинин, окто-аддукт, синтез, идентификация, биотестирование, хлорелла обыкновенная.
but it's application is sufficiently limited by practically complete insolubility and incompatibility with water and water solutions. This belongs also to the most of the light fullerene
1. Dmitriy S. Shevchenko, student of group 3508 Saint Petersburg Electrotechnical University "LETI", e-mail: skarp.ru@yandex.ru Шевченко Дмитрий Сергеевич, студент гр. 3508, СПбГЭТУ «ЛЭТИ»,
2. Olga V. Rakhimova Ph.D (Chem.), Associate Professor St. Petersburg State Electrotechnical University "LETI", e-mail: olga-18061963@yandex.ru
Рахимова Ольга Викторовна, канд. хим. наук, доцент, СПбГЭТУ «ЛЭТИ»
3. Nikolay A. Charykov Dr Sci. (Chem.), Professor, St. Petersburg State Technological Institute (Technical University), e-mail: ncharykov@yandex.ru
Чарыков Николай Александрович д-р хим. наук, профессор, СПбГТИ(ТУ)
4. Konstantin N. Semenov,. Dr Sci. (Chem.), Professor, St. Petersburg State University, e-mail: semenov1986@yandex.ru Семенов Константин Николаевич, д-р хим. наук, профессор, СПбГУ
5. Victor A. Keskinov, Ph.D . (Chem.), Associate Professor, St. Petersburg State Technological Institute (Technical University), e-mail: keskinov@mail.ru
Кескинов Виктор Анатольевич, канд. хим. наук, доцент, СПбГТИ(ТУ)
6. Alexander L. Vorobiev Dr Sci., (Biol.), Professor, D.Serikbayev North Kazakhstan State Technical University
Воробьев Александр Львович, д-р биол. наук, профессор, Восточноказахский государственный технический университет
7. Natalie A. Kulenova, Ph.D (Chem.), Head of the Department of ChMiO, D.Serikbayev North Kazakhstan State Technical University, e-mail: NKulenova@ektu.kz
Куленова Наталья Анатольевна, канд. хим. наук, зав.кафедрой ХМиО, Северокозахстанский государственный технический университет им.Д.Серикбаева,
8. Zhanar S. Onalbaeva, Ph.D (Chem.), Associate Professor, D.Serikbayev North Kazakhstan State Technical University
Оналбаева Жанар Сагидолдиновна, канд. хим. наук, доцент, Северокозахстанский государственный технический университет им.Д.Серикбаева
Дата поступления - 28 августа 2018 года
derivatives (halogen, amino, hydro and others). Meanwhile, water soluble fullerenes may be used in more wide ranges of applications: machinery, building, medicine, pharmacology (as the result of compatibility with water, physiological solutions, blood, lymph, liquor, gastric juice), agriculture, crop production, cosmetics. The adducts of light fullerenes C50 and C70 with amino-acids are the perspective bioactive well water soluble fullerene derivatives. This article continue the cycle of articles, concerning the synthesis, identification and properties investigation of the adducts of light fullerenes and amino-acids [1-18]. Some articles are devoted to the investigation of the influence of water soluble fullerene derivatives on plants growth and development (see, for example [19-22]). In the present article we report about the investigation of octo-adduct of light fullerene C50 with L-arginine C50(C5Hi2NaN4O2)8H8 in bio-testing, using as test microorganism "chlorella vulgaris beijer" - very popular alga for biotesting.
The synthesis and investigation of the adduct of light fullerene Ceo with ar-ginine C6o(C6Hi3N4O2)-sHs.
Synthesis. Arginine hydrochloride (L-C5H14N4O2'HCl) (5 g) and sodium hydroxide (2.5 g) were dissolved in 30 ml of water and 200 ml CH3CH2OH. In the other vessel fullerene C50 (0.5 g) was dissolved in 80 ml o-C5H4(CH3)2. Then both solutions were jointed, mixed and stayed for 120 hours at room temperature. Deep-brown exfoliating solution was formed. "Colorless organic phase" was separated from "inorganic -water one". Water phase was salted out by the excess of methanol (CH3OH) during 24 hours, in this time the sedimentation of the adduct of light fullerene C50 with arginine was completely carried out. The precipitate is filtered and washed repeatedly by the mix of CH3OH with concentrated hydrochloric acid -HCl. Recrystallization of precipitate was carried out triply. Finally precipitate was dried at 50oC for 8 hours. Earlier the original analog of the synthesis R-alanine with C50 adducts was described in the original, to our opinion work [8]. So, the adduct of light fullerene C50 with L-arginine was formed -C50(C5H13N4O2)8H8 (Fig. 1) with the yield ~ 80 % from the theoretically possible. Fig.1
Identification of adduct of light fullerene C60 with L-arginine. Identification of adduct of light fullerene C50 with L-arginine was provided by the following methods: element analysis, infrared and electronic spectroscopy, complex thermal analysis.
Element C-H-O-N analysis. EuroEA3028HT Eurovector Element C-H-O-N analyzator was used. The result of the analysis proved the formula of the adduct of fullerene C50 with argenine - namely C50(C5H13N4O2)8H8. Particularly, correlation between C/N/O in atomic numbers should be: 108/32/15 (or 5.7/2.0/1.0). In this estimation we suppose that the adduct does not contain crystallization water, or the following type crystal hydrate: C50(C5H13N4O2)8H8'nH2O (solid) does not form. In the reality, determined experimental correlation is the following: C/N/O « 5.5/2.0/1.0.
Additionally we should adjudicate that our product contains residual (not washed) quantity of sodium in the correlation C/Na « 100/1. So, finally, refined formula of the adduct between argenine and C50 is the following: C50(C5H13-5Na5N4O2)8H8, where 8 ~ 1. In particular the existence of the residual Na content in the adduct determines comparatively high solubility of the last one in water (see later). Photo of the adduct C50(C5H12NaN4O2)8H8 is represented in Fig. 2.
Fig.2. Photo (optical microscope) of C60(C6H13-SNaN4O2)8H8 crystals (1-100).
Infrared Spectroscopy. A Shimadzu IR Spectrometer IRAffinityl was used in the wavenumbers range v = 450 -4500 cm-1. Solid tablets of C50(C5Hi2NaN4O2)8H8 in dry KBr were used as samples. One can see that longwavelength part
of spectrum: V « 522 - 1715 cm-1, corresponds to the oscillations of C-C bonds in fullerene C50 skeleton , shortwave-
length part of spectrum consists of multiplet: V « 1500 -1700 cm, -1corresponds to the oscillations of C=O and NH2
bonds, V « 3450-3550 cm-1 corresponds to the oscillations of rather free O-H groups.
Transition T, %
4500 1000 3500 3000 2500 2000 1500 1000 500
Wavenumber 1. cm"1
Fig 3. Infrared spectrum of C60(C6H12NaN402)8H8 (KBr tablets).
Electronic Spectroscopy. An Evolution 201 Thermo Fisher spectrophotometer was used for wavelengths C50(C5H12NaN4O2)8H8 against pure water). Fig.4 demonstrates optical density D (l = 1cm) against wavelength (X). One can see that electronic spectrum of C50(C5H13-5NaN4O2)8H8 in visible and near ultraviolet, near infrared region (X = 250 - 1100 nm) is very simple, it has no light absorption peaks and may be characterized by consequently strengthening of light absorption with the lower wavelengths. Additionally one can verify total absence of light absorption peaks of pure non-modified fullerene C50 - for example most intensive one at the wavelength X « 335 nm, the fact proves absence of unreact-ed C50 in the product.
Fig4. Electronic spectrum of water solution of C60(C6H12NaN4O2)8H8 (C = 0.11 g/dm3) against water - D - optical density at l (optical way) = 1 cm, X - wavelength (nm). Bouguer-Lambert-Beerlaw, meanwhile, is fulfilled in the whole spectrum range - Fig. 5. One can use electronic spectrum of in uncharacteristic wavelength range (X = 300^400
nm) to determine volume concentration of C60(C6H13N4O2)8H8
in the solution, for example: C (C60(C6H12NaN4O2)8H8 in g/dm3) = 0.131 D330 (optical way l = 1 cm) (1).
1.4
E 1.2 c
7 P 1.0 °
5 — 0.8
§ § 0.6 ^ f
I 0.4 « 02
o.o
0.00 0.05 0,10 0,15 0,20 0,25
Concentration the adduct of C^ with argcninc - C (g/dm )
Fig. 5. The dependence of optical density of water solution of C60(C6H12NaN402)8H8 against water - D - optical density at l (op-
2
tical way) = 1 cm, ^ - wavelength = 330 nm.
Complex thermal analysis of crystal
C60(C6H12NaN4O2)8H8 Complex thermal analysis of C60(C6H12NaN402)8H8, equilibrium to saturated water solution at room temperature, was executed in the temperature range 20-650 °C. Thermo-gravimeter NETZSCH STA 449F3 was used (velocity of the analysis v « 2 K/min, atmosphere -air). Results are represented in Fig. 6. Analogous behavior with multistage consequent dehydration and decarboxylation we have observed earlier for hydrate of C60[(=C(C00H)2]3 [12]. One can see the following:
- No crystal hydrates in the solid phase -C50(C5H12NaN402)8H8-nH20 are formed.
- In the temperature range T~100^530 °C multistage consequent processes of destroying of functional "argenine groups" - C6H12NaN402, in the presence of 02 (air) is realized, these processes are accompanied by oxidation: dehydration (-nH20), soft decarboxylation (-nC0), diazotizing (-N2), dehy-drogenation(-H2) . In the end of these processes at the temperature T ~ 520^540 °C practically complete deletion of argenine groups occurs, that would be equivalent to the loss of the initial mass (m0) - [Am/m0]-100 % = 69 rel. %.
- At higher temperatures T~530^650 °C oxidizing processes of the destroying of fullerene core (C60) start.
- It may be interesting, that DTG (differential mass curve dm/dT(T)) is almost non-informative, whereas TG - m(T) and DSC -
Bio-testing of light fullerene Ceo with L-arginine Ceo(CeHi2NaN4O2)8H8.
Methodic. During the study of the properties of adduct fullerene C60 experiments were conducted to verify the toxicity of aqueous solutions of this compound by biotesting. As a test micro-organism, a laboratory-pure strain of algae chlorella vulgaris bijer was selected.
The method of the experiment consists in the periodic recording of changes in the optical density of the solution under study, which consists of a nutrient medium with a suspension of algae and an aqueous adduct solution, in a ratio of 3/1 [23, 24]. All changes in the optical density of the solution are associated only with an increase in the biomass of the algae colony in the sample. After sample preparation, the initial values of optical densities at a wavelength A=660 nm for all samples including the control were recorded by the Unico 2800 spectrophotometer. For the accelerate the cultivation of algae was used bioreactor, collected on the basis of thermostatic water bath TJ-TB-01 and led lamp with two emitters (red A= 650-660 nm, blue A= 440-450 nm.). These wavelengths correspond to the maximum of light absorption of plant photosystems, which are responsible for photosynthesis and chemosynthesis, the graph of dependence is shown in Fig. 7 (data from [25]).
In the framework of the experiment we used an aqueous solution of arginine octo-adduct of fullerene C60 with the concentration of 0.5 g/l, further, from the sample of this solution was prepared diluted solutions with the dilution on the ratio of 1/1, 1/10, 1/100. In the reactor was also placed a control sample (Cont) which contained only culture medium and a suspension of algae chlorella vulgaris beijer. Cultivation was carried out for 3 hours, the average values were obtained during parallel 2 experiments with the same concentrations, the results are represented in Table. The obtained data were averaged, on the basis of which a graph of the dependence of the population growth in time was constructed (Fig. 8). Data are expressed in optical densities at wavelength 660 nm and optical way l = 1 cm (a.u.). Values are directly proportional to chlorella vulgaris bijer volume concentrations.
Fig.7. The dependencies of light absorption activity in the different processes ofphotosynthesis and chemosynthesis (blue - chlorophyll synthesis, green - photosynthesis, red - photo-morphogenesis) on the wavelength in visional region of light wavelength
dQ/dT(T) are fit to interpretation.
Fig 6. Complex thermal analysis ofcrystal C60(C6H12NaN402)8H8.
Table. The results of cultivation of chlorella vulgaris bijer at different dilutions
Probe dilution
0 hours
D550 - 0ptical density at wavelength 660 nm and optical way l = 1 cm (a.u.) Values are directly proportional to chlorella vulgaris bijer volume concentrations
Average
1 hour
Ave-rage
2 hours
Average
3 hours
Average
Cont 1/1 1/10 1/100
0.104 0.348 0.125 0.099
0.100 0.324 0.132 0.108
0.102 0.336 0.129 0.104
0.115 0.390 0.160 0.117
0.121 0.397 0.175 0.124
0.118 0.394 0.168 0.121
0.130 0.448 0.168 0.122
0.132 0.429 0.184 0.130
0.131 0.439 0.176 0.126
0.117 0.440 0.15 0.126
0.111 0.429 0.178 0.126
0.114 0.435 0.164 0.126
On the basis of the experimental data obtained, it can be concluded that arginine octo-adduct of fullerene C60 with dilution of 1/1 and 1/10 had a significant effect on the rate of growth of algae chlorella vulgaris bijer, the value of the number of algae in the sample with dilution 1/1 contents above 3.5 times than in the control sample, even in the second hour of the experiment. Therefore, it can be concluded that this substance has the property of accelerating the growth of algae colony biomass, the toxic effect, at this concentration, on the test organism chlorella vulgaris bijer - is absent. Visual comparison size of population at the dilution 1/1 and Control sample is represented in Fig. 9.
1 1,5 2
Time of experiment (hours) Control — Dilution 1:1 — Dilution 1:10 — Dilution 1:100
Fig. 8. The dependencies of growth of chlorella vulgaris bijer population (rel.units) on the time of cultivation at the different dilutions: blue - Control, violet - dilution 1/100, green - dilution 1/10, red -dilution 1/1.
Fig.9. Visual comparison of size of population at the dilution 1/1 (right) and Control sample (left).
Conclusions
On the basis of the experimental data obtained, it can be concluded that arginine octo-adduct of fullerene C60 with dilution of original water solution 1/1 and 1/10 (2.5 and 0.25 g/dm3) had a significant effect on the rate of growth of algae chlorella vulgaris bijer, the value of the number of algae in the sample with dilution 1/1 contents above 3.5 times than in the control sampl5e, even in the second hour of the experiment. Therefore, it can be concluded that this substance has the property of accelerating the growth of algae colony biomass, the toxic effect, at this concentration, on the test organism chlorella vulgaris bijer - is absent.
Acknowledgements
This work was supported by Russian Foundation of Fundamental Investigations - RFFI (Projects № 16-08-01206, 1808-00143).
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