Лаборатория химии липидов

Руководитель: Водовозова Елена Львовна, д. х. н.
+7 (495) 330-66-10 · Elvod@ibch.ru

lipids.ibch.ru

Лаборатория химии липидов была создана в 1963 г. по инициативе академика М. М. Шемякина; первым ее возглавил д. х. н. (позднее профессор, затем член-корреспондент АН СССР) Л. Д. Бергельсон. С 1991 по 2007 г. лабораторией руководил д. х. н. Юл. Г. Молотковский; с 2008 г. руководитель — д. х. н. Е. Л. Водовозова.

Направления исследований

В настоящее время в лаборатории развиваются два основных направления исследований:

  • Создание наноразмерных систем направленной доставки лекарств на основе липосом, липофильных пролекарств и липофильных гликоконъюгатов (молекулярных адресов) (рис. 1, 2). Показано, что адресные лекарственные липосомы по противоопухолевому эффекту значительно превосходят исходные лекарства и липосомы, не оснащенные углеводным лигандом (рис. 3).
  • Синтез новых флуоресцентных зондов липидной природы, изучение с их помощью строения и функций мембран и закономерностей переноса энергии возбуждения между флуорофорами (рис. 4 и 5).

Основные достижения

За прошедшие годы в лаборатории выполнен ряд важных исследований в области биоорганической химии, биохимии, биофизики и медицинской химии, вызвавших широкий интерес и получивших признание.

Среди этих работ: изучение стереохимии реакции Виттига и синтез на ее основе ряда природных ненасыщенных жирных кислот (Л. Д. Бергельсон и В. А. Вавер); обнаружение явления дедифференцировки липидного состава органелл раковых клеток (Л. Д. Бергельсон и Э. В. Дятловицкая); широкое исследование распространения и роли нового класса диольных липидов (Л. Д. Бергельсон и В. А. Вавер); синтез и доказательство строения бактериальных липоаминокилот, а также первый синтез ненасыщенного фосфатидилинозита (Л. Д. Бергельсон и Ю. Г. Молотковский); изучение топологии мембран с помощью спектроскопии ЯМР (Л. Д. Бергельсон и Л. И. Барсуков ― в сотрудничестве с В. Ф. Быстровым); обнаружение и доказательство строения нового класса бактериальных орнитин-содержащих липидов (Л. Д. Бергельсон и С. Г. Батраков); синтез и применение в биологических исследованиях большой гаммы флуоресцентных и фотоаффинных липидных зондов (Л. Д. Бергельсон, Ю. Г. Молотковский, Е. Л. Водовозова, И. И. Михалев, И. А. Болдырев); разработка адресной липосомной доставки в опухоли липид-модифицированных противораковых агентов (Ю. Г. Молотковский, Е. Л. Водовозова, Г.П. Гаенко ― в сотрудничестве с Н. В. Бовиным).

В настоящее время в лаборатории развиваются два основных направления исследований. Первое ― создание наноразмерных систем направленной доставки лекарств на основе липосом, липофильных пролекарств и липофильных гликоконъюгатов (молекулярных адресов) (рисунки 1 и 2). Показано, что адресные лекарственные липосомы по противоопухолевому эффекту значительно превосходят исходные лекарства и липосомы, не оснащенные углеводным лигандом (рис. 3). Второе направление исследований ― синтез новых флуоресцентных зондов липидной природы, изучение с их помощью строения и функций мембран и закономерностей переноса энергии возбуждения между флуорофорами (рисунки 4 и 5).

Рис. 1. Структуры липофильных пролекарств и гликоконъюгатов.

Рис. 2. Электронные микрофотографии реплик с поверхностей скола замороженных дисперсий: А, а — липосом c МТХ-DG;Б, б — липосом c Mlph-DG.

Рис. 3. Динамика выживания мышей BLRB с перевитой аденокарциномой молочной железы в различных экспериментальных группах (n=10). Мыши получили в/в инъекции на 3-й и 7-й день после перевивки опухоли. Группы: 1 — мерфалан (сарколизин); 2 — пустые липосомы; 3 — липосомы + пролекарство; 4 — липосомы + пролекарство + конъюгат SiaLeX; 5 — липосомы + конъюгат SiaLeX; контроль —  физ. раствор.

Рис. 4. Набор флуоресцентных зондов для исследования свойств мембран на разном расстоянии от поверхности мембраны. Графики на заднем плане — профили параметра порядка набора зондов в бислоях разного состава.

lipchem

Рис. 5. Структура ганглиозидного зонда BODIPY–FL–C3–GM1 — флуоресцентного маркера рафтов.

Ф.И.О.ДолжностьЭл. почта
Алексеева Анна инж.-иссл.
Болдырев Иван Александрович, к. х. н.н.с.
Вострова Анна Григорьевна, к. х. н.н.с.
Гаенко Галина Петровна, к. б. н.с.н.с.
Жедулов Александр Евгеньевичм.н.с.
Жукова Галина Ивановнатех.-лаб.
Кузнецова Наталья Ростиславовнаасп.
Михалёв Илья Ильич, к. х. н.с.н.с.
Молотковский Юлиан Георгиевич, д. х. н., профессорг.н.с.
Павлова Юлия Борисовнаинж.-иссл.

Избранные публикации

  1. Sachl R., Boldyrev I., Johansson L.B.A. (2010). Localisation of BODIPY-labelled phosphatidylcholines in lipid bilayers. Phys. Chem. Chem. Phys. , [+]

    A series of sn-2 acyl-labelled phosphatidyl-cholines (PC), bearing 4,4-difluoro-1-3-5-7-tetra-methyl-4-bora-3a,4a-diaza-s-indacene-8-yl (Me4-BODIPY) at the end of the Cn-acyl chains were solubilised in unilamellar vesicles and studied with respect to the order and location of the Me4-BODIPY (denoted: B) group. The obtained results are based on time-resolved electronic energy transfer from donors (2-(9-anthroyloxy)-stearic acid) localised in the lipid–water interface to acceptors BnPC (n = 3, 5, 7, 9, 11, 13, 15), as well as the energy migration among the Me4-BODIPY groups of BnPC:s. The donor–acceptor and the donor–donor experiments strongly suggest that the Me4-BODIPY group in BnPC tends to loop back close to the lipid–water interface. The Me4-BODIPY groups, residing in the two bilayer leaflets, are located at approximately the same depth, and transversally separated by ca. 27 Å for all n-values. Close to the interface, the optimal transversal distribution widens somewhat with increasing length of the sn-2 acyl chain. The obtained order parameter profile of the BnPC:s is also compatible with such a location.

  2. Mikhalyov I., Gretskaya N., Johansson L.B. (2009). Fluorescent BODIPY-labelled GM1 gangliosides designed for exploring lipid membrane properties and specific membrane-target interactions. Chem. Phys. Lipids 159 (1), 38–44 [+]

    New fluorophore-labelled G(M1) gangliosides have been synthesised and spectroscopically characterised. Spectroscopically different BODIPY groups were covalently linked, specifically to either the polar or the hydrophobic part of the ganglioside molecule. The absorption and fluorescence spectroscopic properties are reported for 564/571-BODIPY- and 581/591-BODIPY-labelled G(M1). Each of the different BODIPY groups is highly fluorescent and depolarisation experiments provide molecular information about the spatial distribution in lipid bilayers, as well as order and dynamics. From experiments performed on two spectroscopically different BODIPY:s, specific interactions can be revealed by monitoring the rate/efficiency of donor-acceptor electronic energy transfer. Systems of particular interest for applying these probes are e.g. mixtures of lipids, and peptides/proteins interacting with lipid membranes.

  3. Kuznetsova N., Kandyba A., Vostrov I., Kadykov V., Gaenko G., Molotkovsky J., Vodovozova E. (2009). Liposomes loaded with lipophilic prodrugs of methotrexate and melphalan as convenient drug delivery vehicles. J. Drug. Deliv. Sci. Techn. 19, 51–59 [+]

    Liposomal formulations prepared by extrusion from natural phospholipids and 1,2-dioleoylglycerol conjugates of methotrexate and melphalan (egg phosphatidylcholine–phosphatidylinositol–prodrug, 8:1:1, by mol.) were characterized by size, composition and stability. Both prodrugs were shown to incorporate completely into unilamellar liposomes with the mean size below 100 nm and form stable dispersions containing the drug concentrations relevant for systemic injections in animals. For long-term storage, the dispersions can be  subjected to deep freezing (- 196°C) and stored at - 70°C; before usage, they should be defrosted and treated shortly in an ultrasonic bath. According to the example of methotrexate conjugate, stability of prodrug ester bond in liposomal formulation towards hydrolysis by human plasma esterases during 24-h incubation were established. Also, liposomes bearing methotrexate conjugate were shown to overcome resistance of human leukemia cells related to impaired transport of initial drug across the membrane.

  4. Vodovozova E.L., Pazynina G.V., Tuzikov A.B., Grechishnikova I.V., Molotkovsky J.G. (2009). Synthesis of photoreactive inorganic probes--instruments for studying membrane lectins. Bioorg. Khim. 30 (2), 174–81 [+]

    A method for the synthesis of photoaffinity neoglycolipid probes with a highly efficient carbene-generating diazocyclopentadien-2-ylcarbonyl (Dcp) label, which can be radioiodinated under standard oxidation conditions, was developed. The probes are intended for incorporation into the lipid bilayer. They are lipophilic glycoconjugates on the basis of an amphiphilic aglycone built up from a diacylglycerol and a polyethylene glycol spacer (with a polymerization degree of 9-16) bearing the Dcp label at the terminal unit. The location of the label in the aglycone provides the possibility of one-step preparation of a wide range of probes using various carbohydrate synthons. We have synthesized photoaffinity neoglycoconjugates containing the oligosaccharides: sialyl LewisX tetrasaccharide and A trisaccharide, which is specific to some tumor cells. A probe containing an inactive pentaol (aminodeoxyglucitol) was also synthesized to detect nonspecific binding. The Dcp label is bound to the probe molecule by ester bond; its lability under alkaline conditions facilitates the analysis of cross-linked products after photoaffinity labeling. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 2; see also http://www.maik.ru.

  5. Boldyrev I.A., Zhai X., Momsen M.M., Brockman H.L., Brown R.E., Molotkovsky J.G. (2007). New BODIPY lipid probes for fluorescence studies of membranes. J. Lipid Res. 48 (7), 1518–32 [+]

    Many fluorescent lipid probes tend to loop back to the membrane interface when attached to a lipid acyl chain rather than embedding deeply into the bilayer. To achieve maximum embedding of BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) fluorophore into the bilayer apolar region, a series of sn-2 acyl-labeled phosphatidylcholines was synthesized bearing 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-yl (Me(4)-BODIPY-8) at the end of C(3)-, C(5)-, C(7)-, or C(9)-acyl. A strategy was used of symmetrically dispersing the methyl groups at BODIPY ring positions 1, 3, 5, and 7 to decrease fluorophore polarity. Iodide quenching of the phosphatidylcholine probes in bilayer vesicles confirmed that the Me(4)-BODIPY-8 fluorophore was embedded in the bilayer. Parallax analysis of Me(4)-BODIPY-8 fluorescence quenching by phosphatidylcholines containing iodide at different positions along the sn-2 acyl chain indicated that the penetration depth of Me(4)-BODIPY-8 into the bilayer was determined by the length of the linking acyl chain. Evaluation using monolayers showed minimal perturbation of <10 mol% probe in fluid-phase and cholesterol-enriched phosphatidylcholine. Spectral characterization in monolayers and bilayers confirmed the retention of many features of other BODIPY derivatives (i.e., absorption and emission wavelength maxima near 498 nm and approximately 506-515 nm) but also showed the absence of the 620-630 nm peak associated with BODIPY dimer fluorescence and the presence of a 570 nm emission shoulder at high Me(4)-BODIPY-8 surface concentrations. We conclude that the new probes should have versatile utility in membrane studies, especially when precise location of the reporter group is needed.

  6. Boldyrev I.A., Molotkovskiĭ I.G. (2006). [A synthesis and properties of new 4,4-difluoro-3a,4a-diaza-s-indacene (BODIPY))-labeled lipids]. Bioorg. Khim. 32 (1), 87–92 [+]

    A series of fluorescently labeled fatty acids of various chain lengths with 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-yl (Me4-BODIPY-8) residue in the omega-position were synthesized. These acids were used to prepare new fluorescently labeled phosphatidylcholines, sphingomyelin, and galactosyl ceramide. Taking into account the symmetry of the Me4-BODIPY-8-fluorophore, one can presume that, in most bilayer membrane systems, this fluorophore is would be embedded into the bilayer.

  7. Boldyrev I.A., Molotkovskiĭ Yu.G. (2005). A synthesis of new rigid fluorescent bichromophoric probes for studying mechanisms of donor-donor energy migration. Bioorg. Khim. 31 (3), 331–4 [+]

    Three new fluorescent probes were synthesized for improving the method of studying donor-donor energy migration (DDEM). Each probe has two identical fluorescent 7-diethylaminocoumarin-3-carbonyl groups attached to a rigid bisteroid dodecacyclic spacer through additional inserts. In two probes, the inserts are beta-Ala and L-Ser residues, which provide for a different nearest environment of the fluorophores. The third probe has identical beta-Ala inserts. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 3; see also http://www.maik.ru.

  8. Boldyrev I.A., Molotkovskiĭ Yu.G. (2004). Fluorescent properties of 9-anthracenecarboxamides. Bioorg. Khim. 30 (6), 649–55 [+]

    A number of new 9-anthracenecarboxamides are synthesized in order to create new fluorescent probes for studying biological systems. The parameters of their fluorescence in organic solvents of various polarities are investigated, and possible mechanisms of internal quenching of fluorescence of these compounds are discussed. One of the compounds, 4-ethoxycarbonylphenylamide of 9-anthracenecarboxylic acid, is shown to be a promising basis for the development of a new fluorescent probe. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 6; see also http://www.maik.ru.

  9. Vodovozova E.L., Tsibizova E.V., Molotkovsky J.G. (2001). One-step iodination of the diazocyclopentadien-2-ylcarbonyl group—a new and convenient preparation of effective radiolabelled photoaffinity probes. J. Chem. Soc., Perkin Trans. 1  (200), 2221–2228 [+]

    A detailed study devoted to direct iodination of the photoactivatable diazocyclopentadien-2-ylcarbonyl (Dcp) group is presented. The iodination does not influence the high carbene reactivity of the Dcp-generated carbene. It was shown that the Dcp substituent forms 4-mono-, 5-mono- and 4,5-diiododerivatives upon iodination under oxidative conditions (76, 20 and 4%, respectively, when DcpOMe 2 is iodinated). Photolysis of the individual products of iodination in cyclohexane resulted in rather high insertion into non-activated CH bonds, without noticeable loss of iodine. Syntheses of new phospholipid and ganglioside membrane probes are also described which incorporate the Dcp function via a labile ester bond. A [125I]-Dcp-phosphatidylcholine probe exhibiting high specific radioactivity (∼500 Ci mmol1) was easily prepared at yields of 90% (on the starting Na125I), by using peracetic acid as an oxidant.
    Furthermore, it was successfully used for photolabelling of the integral protein hemagglutinin in a well-characterised influenza virus model. In summary, the Dcp group is efficient for labelling a wide variety of molecules, and as such, it provides a new tool for exploring a diverse range of biological systems.

  10. Vodovozova E.L., Moiseeva E.V., Grechko G.K., Gayenko G.P., Nifant'ev N.E., Bovin N.V., Molotkovsky J.G. (2000). Antitumour activity of cytotoxic liposomes equipped with selectin ligand SiaLe(X), in a mouse mammary adenocarcinoma model. Eur. J. Cancer 36 (7), 942–9 [+]

    The overexpression of lectins by malignant cells compared with normal ones can be used for the targeting of drug-loaded liposomes to tumours with the help of specific carbohydrate ligands (vectors). Recently we have shown that liposomes bearing specific lipid-anchored glycoconjugates on a polymeric matrix bind in vitro to human malignant cells more effectively and, being loaded with a lipophilic prodrug of merphalan, reveal higher cytotoxic activity compared with unvectored liposomes. In this study, carbohydrate-equipped cytotoxic liposomes were tested in vivo in a mouse breast cancer model, BLRB-Rb (8.17)1Iem strain with a high incidence of spontaneous mammary adenocarcinoma (SMA). Firstly, a cell line of the SMA was established which was then used to determine the specificity of the tumour cell lectins. After screening of the lectin specificity of a number of fluorescent carbohydrate probes, SiaLe(X) was shown to be the ligand with the most affinity, and a lipophilic vector bearing this saccharide was synthesised. Then different liposomal formulations of the synthetic merphalan lipid derivative and SiaLe(X) vector were prepared and applied in the treatment of mice with grafted adenocarcinomas. The results of the tumorigenesis data show that the therapeutic efficacy of merphalan increases sharply after its insertion as a lipophilic prodrug into the membrane of SiaLe(X)-vectored liposomes.

  11. Vodovozova E.L., Gayenko G.P., Razinkov V.I., Korchagina E.Y., Bovin N.V., Molotkovsky J.G. (1998). Saccharide-assisted delivery of cytotoxic liposomes to human malignant cells. Biochem. Mol. Biol. Int. 44 (3), 543–53 [+]

    The overexpression of lectins by malignant cells was applied for in vitro targeting of liposomes equipped with a saccharide vector and loaded in the lipid phase with a lipid derivative of anticancer agent sarcolysine. The lectin specificity of human leukemia HL-60 and human lung adenocarcinoma ACL cells was revealed by tests with fluorescein-labeled sugar probes. With the help of fluorescent lipid dye it was shown that active saccharide ligands increased the level of the vectored liposome binding to malignant cells by 50-80% as compared to liposomes without vector or with inactive one. The degree of liposome/cell membrane fusion was monitored fluorometrically and was shown to be complete and independent of the vectors. The targeted drug-loaded liposomes had the cytotoxic activity 2-4 times higher as compared to the vector-free ones.

  12. Batrakov S.G., Bergelson L.D. (1978). Lipids of the Streptomycettes. Structural investigation and biological interrelation. Review. Chem. Phys. Lipids 21 (1-2), 1–29 [+]

    During a systematic investigation of lipids of Streptomycetes a series of compounds of biochemical and microbiological interest have been isolated and characterized. These include several menaquinones, glycosyl diglycerides (glucuronosyl and isoladobinosym diglycerides), two ornithino lipids and a diol phospholipid. Some of these lipids were not known previously as constituents of streptomycete cells although they have been encountered elsewhere; others have proved to be novel lipids. The results of structural studies of these lipids are reviewed and some of their possible biological functions are discussed.

  13. Molotkovsky J.G., Bergelson L.D. (1973). Synthesis of unsaturated mixed acid phosphatidylinositol of natural configuration. A new procedure for resolving racemic alcohols.  (11), 135–147
  14. Bergelson L.D., Dyatlovitskaya E.V., Torkhovskaya T.I., Sorokina I.B., Gorkova N.P. (1968). Dedifferentiation of phospholipid composition in subcellular particles of cancer cells. FEBS Lett. 2 (2), 87–90 [+]

    In this pioneer work, the phenomenon of phospholipid dedifferentiation (composition leveling) in the tumor cell organelles was described for the first time.

  15. Bergelson L.D., Vaver V.A., Prokazova N.V., Ushakov A.N., Popkova G.A. (1966). Diol lipids. Biochim. Biophys. Acta 116 (3), 511–20 [+]

    In the first review on the recently discovered diol lipids, the data on their structures and distribution in natural sources are summarized, and a hypothesis of their metabolic role is expressed.

Руководитель подразделения

Водовозова Елена Львовна

  • Москва, ул. Миклухо-Маклая, 16/10 — На карте
  • ИБХ РАН, корп. 34, комн. 532
  • Тел.: +7 (495) 330-66-10
  • Эл. почта: Elvod@ibch.ru