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).

Ф.И.О.	Должность	Эл. почта
Алексеева Анна Сергеевна	инж.-иссл.	anna@lipids.ibch.ru
Болдырев Иван Александрович, к. х. н.	н.с.	ivan@lipids.ibch.ru
Вострова Анна Григорьевна, к. х. н.	н.с.	anna.vostrova@gmail.com
Гаенко Галина Петровна, к. б. н.	с.н.с.	GPG008@mail.ru
Жукова Галина Ивановна	тех.-лаб.
Кузнецова Наталья Ростиславовна	м.н.с.	natalia@lipids.ibch.ru
Михалёв Илья Ильич, к. х. н.	с.н.с.	Ilya.Mikhalyov@gmail.com
Молотковский Юлиан Георгиевич, д. х. н., профессор	г.н.с.	jgmol@ibch.ru

Избранные публикации

1. Kuznetsova N.R., Sevrin C., Lespineux D., Bovin N.V., Vodovozova E.L., Mészáros T., Szebeni J., Grandfils C. (2011). Hemocompatibility of liposomes loaded with lipophilic prodrugs of methotrexate and melphalan in the lipid bilayer. Journal of controlled release : official journal of the Controlled Release Society , [+]

   A panel of in vitro tests intended for evaluation of the nano-sized drug delivery systems' compliance with human blood was applied to liposomal formulations of anticancer lipophilic prodrugs incorporated into the lipid bilayer. Liposomes on the basis of natural phosphatidylcholine (PC) and phosphatidylinositol (PI), 8:1 (mol) were loaded with 10mol% of either methotrexate or melphalan 1,2-dioleoylglyceride esters (MTX-DOG and Mlph-DOG respectively) and either decorated with 2mol% of sialyl Lewis X/A (SiaLe(X/A)) tetrasaccharide ligand or not. Hemolysis rate, red blood cells and platelets integrity and size distribution, complement (C) activation, and coagulation cascade functioning were analyzed upon the material incubation with whole blood. Both formulations were negatively charged with the zeta potential value being higher in the case of MTX-DOG liposomes, which also were larger than Mlph-DOG liposomes and more prone to aggregation. Accordingly, in hemocompatibility tests Mlph-DOG liposomes did not provoke any undesirable effects, while MTX-DOG liposomes induced significant C activation and abnormal coagulation times in a concentration-dependent manner. Reactivity of the liposome surface was not affected by the presence of SiaLe(X/A) or PI. Decrease in liposome loading with MTX-DOG from 10 to 2.5% resulted in lower surface charge density, smaller liposome size and considerably reduced impact on C activation and coagulation cascades.

2. Semenova A.A., Chugunov A.O., Dubovskii P.V., Chupin V.V., Volynsky P.E., Boldyrev I.A. (2011). The role of chain rigidity in lipid self-association: Comparative study of dihexanoyl- and disorbyl-phosphatidylcholines. Chem. Phys. Lipids , [+]

   In the course of structure-function investigations of lipids a phosphatidylcholine molecule with short and rigid tails, di-2,4-hexadienoylphosphatidylcholine (DiSorbPC), was synthesized and studied in comparison with its saturated analog, dihexanoylphosphatidylcholine (DHPC). Conjugated double bonds in the acyl chains in DiSorbPC reduce considerably the number of possible conformers of the lipid within an aggregate. This leads to impaired packing of unsaturated acyl chains and thus, to a surprisingly high (115Å(2)) area per molecule for DiSorbPC at the air-water interface and failure to form micelles of regular size and shape. Details on DiSorbPC aggregation and packing provided by a set of experimental techniques combined with molecular dynamics simulations are presented.

3. Alekseeva A.S., Maslov M.A., Antipova N.V., Boldyrev I.A. (2011). Comparison of two lipid/DNA complexes of equal composition and different morphology. Colloids Surf B Biointerfaces 88 (1), 512–6 [+]

   Two types of complexes were prepared from a cationic cholesterol derivative, dioleoylphos-phatidylcholine and DNA. Depending on the preparation procedure complexes were either dense snarls of lipid covered DNA (type A) or multilayer liposomes with DNA between layers (type B). The transfection efficiency of the snarl-shaped complexes was low but positive. The transfection efficiency of the liposome-shaped complexes was zero, while DNA release upon their interaction with anionic liposomes was 1.7 times higher. The differences in transfection efficacy and DNA release could not be ascribed to the difference in resistance of complexes to decomposition upon interaction with anionic liposomes or intracellular environment since the lipid composition of complexes is the same. Instead the complexes in which lipoplex phase is more continuous (type A) should require more anionic lipids or more time within a cell for complete decomposition. Prolonged life time should lead to the higher probability of DNA expression.

4. Moiseeva E.V., Kuznetsova N.R., Svirshchevskaya E.V., Bovin N.V., Sitnikov N.S., Shavyrin A.S., Beletskaya I.P., Combes S., Fedorov A.Y.u., Vodovozova E.L. (2011). Liposome formulations of combretastatin A4 and its 4-arylcoumarin analogue prodrugs: The antitumor effect in the mouse model of breast cancer. Biochemistry (Moscow) Supplement Series B: Biomedical Chemistry Biochemistry (Moscow) Supplement Series B: Biomedical Chemistry 5 (3), 276–283 [+]

   The antimitotic agent combretastatin A-4 (CA-4) has been recently proposed as an antivascular agent for anticancer therapy. In order to reduce systemic toxicity by means of administration in liposome formulations, new lipophilic prodrugs, oleic derivatives of CA-4 and its 4-arylcoumarin analogue (CA4-Ole and ArC-Ole, respectively), have been synthesized in this study. Liposomes with mean diameter of 100 nm prepared on the basis of egg phosphatidylcholine and baker’s yeast phosphatidylinositol quantitatively included up to 15 mol% of CA4-Ole, or 7 mol% of ArC-Ole. To achieve targeting to neovascular endothelium prodrug bearing liposomes decorated with the tetrasaccharide selectin ligand Sialyl Lewis X (SiaLeX) have been also prepared. The antitumor activity was studied in vivo using the model of slow-growing mouse breast cancer. Under the dose used (22 mg/kg) and the administration protocol (four injections, one per a week, starting from the appearance of palpable tumors) cytostatic CA-4 did not reveal any anticancer effect; moreover, it even stimulated tumor growth. The liposome formulations of CA4-Ole did not demonstrate such stimulation. However, to achieve a pronounced antitumor effect, the number of injections of liposomes should be apparently increased. The cytotoxic activity of a novel antimitotic agent ArC was one order of magnitude lower in the human breast carcinoma cell culture in vitro. Nevertheless, in vivo in the mouse model of breast cancer the antitumor effect of this compound corresponded to the double equivalent dose of CA-4. The results demonstrate perspectives of SiaLe^X-liposomes loaded with ArC-Ole: the preparation partially inhibited tumor growth already after the second injection. Thus, subsequent optimization of doses and regimens of administration both for ArC and liposomal ArC-Ole formulations are needed.

5. Mikhalyov I., Samsonov A. (2011). Lipid raft detecting in membranes of live erythrocytes. Biochim. Biophys. Acta 1808 (7), 1930–9 [+]

   The fluorescent probe N-(BODIPY(®)-FL-propionyl)-neuraminosyl-GM(1) (BODIPY-GM(1)) was used to detect lipid rafts in living red blood cells (RBCs) membranes. The probe was detected with fluorescence video microscopy and was found to be uniformly distributed along plasma membrane at room temperature (23°C). At 4°C some probe clearly phase-separated to yield detectable bright spots that were smaller than spatial resolution. As measured by spectrofluorometry, in addition to a major fluorescence peak caused by emissions from monomers, the probe exhibited a red-shifted peak that is characteristic of a BODIPY fluorophore at high local concentrations, indicating that some probe had clustered. Red-shifted fluorescence was the greatest at 4°C, intermediate at 23°C, and the smallest at 37°C. Treating the RBCs with methyl-β-cyclodextrin to remove cholesterol eliminated the red-shifted peak. This strongly indicates that the presence of cholesterol was essential for phase separation of the probe. Fluorometry experiments indicate that rafts exist at 23°C and at 37°C, even though the membrane appears to be uniform at the resolution of microscope. The distinct GM(1) patches distributed over entire membrane of the erythrocytes were observed at both 23°C and at 37°C in RBCs stained with Alexa FL 647 cholera toxin subunit B conjugate (CTB-A647 ). Based on both fluorometry and fluorescence microscopy, some rafts clearly exist at 37°C.

6. Sachl R., Mikhalyov I., Gretskaya N., Olżyńska A., Hof M., Johansson L.B. (2011). Distribution of BODIPY-labelled phosphatidylethanolamines in lipid bilayers exhibiting different curvatures. Phys Chem Chem Phys 13 (24), 11694–701 [+]

   In this paper we have investigated the behaviour of newly synthesised mono-palmitoyl- and dipalmitoyl-phosphatidylethanolamine probes (abbreviated as mPE and dPE, respectively) labelled in the polar headgroup region by either the FL-BODIPY or the 564/570-BODIPY fluorophore and solubilised in lipid systems that exhibit different curvatures. Because of the bulky BODIPY-groups, the monoacyl-form derivatives have a conic-like shape, whereas that for the diacyl derivatives is rather cylindrical. A careful analysis of time-resolved resonance energy transfer experiments by means of analytical models as well as Monte Carlo simulations shows that the mPE derivatives have a comparable affinity to highly curved bilayer regions (torroidal pores formed by magainin-2 in lipid bilayers, or the rims of discoid bicelles) and to planar bilayer regions (i.e. the flat region of lipid bilayers and bicelles). Furthermore, the monoacyl-probes are as compared to the diacyl-probes effectively closer to each other in a lipid bilayer, while none of these probes seems to be randomly distributed. Self-aggregation is most efficiently induced by the larger aromatic 564/570-BODIPY chromophore, but it is suppressed when using the diacyl instead of the monoacyl-form, and/or by attaching BODIPY-groups to the acyl-chain.

7. Vodovozova E.L., Pazynina G.V., Bovin N.V. (2011). Synthesis of diglyceride conjugate of selectin ligand SiaLeX as a vector for targeting of drug-loaded liposomes. Mendeleev Communications 21 (2), 69–71 [+]

   A conjugate of tetrasaccharide Sialyl Lewis X [SiaLe^X, Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcβ] 3-aminopropyl glycoside and rac-1,2-dioleoyl-3-carboxymethylene[poly(8–15)oxyethylene]oxyacetylamidopropionylglycerol amenable for the incorporation in lipid bilayer of drug-loaded liposomes to achieve targeting in tumors and inflammation foci was obtained by the formation of carboxamide bond.

8. Konshina A.G., Boldyrev I.A., Utkin Y.N., Omelkov A.V., Efremov R.G. (2011). Snake cytotoxins bind to membranes via interactions with phosphatidylserine head groups of lipids. PLoS ONE 6 (4), e19064 [+]

   The major representatives of Elapidae snake venom, cytotoxins (CTs), share similar three-fingered fold and exert diverse range of biological activities against various cell types. CT-induced cell death starts from the membrane recognition process, whose molecular details remain unclear. It is known, however, that the presence of anionic lipids in cell membranes is one of the important factors determining CT-membrane binding. In this work, we therefore investigated specific interactions between one of the most abundant of such lipids, phosphatidylserine (PS), and CT 4 of Naja kaouthia using a combined, experimental and modeling, approach. It was shown that incorporation of PS into zwitterionic liposomes greatly increased the membrane-damaging activity of CT 4 measured by the release of the liposome-entrapped calcein fluorescent dye. The CT-induced leakage rate depends on the PS concentration with a maximum at approximately 20% PS. Interestingly, the effects observed for PS were much more pronounced than those measured for another anionic lipid, sulfatide. To delineate the potential PS binding sites on CT 4 and estimate their relative affinities, a series of computer simulations was performed for the systems containing the head group of PS and different spatial models of CT 4 in aqueous solution and in an implicit membrane. This was done using an original hybrid computational protocol implementing docking, Monte Carlo and molecular dynamics simulations. As a result, at least three putative PS-binding sites with different affinities to PS molecule were delineated. Being located in different parts of the CT molecule, these anion-binding sites can potentially facilitate and modulate the multi-step process of the toxin insertion into lipid bilayers. This feature together with the diverse binding affinities of the sites to a wide variety of anionic targets on the membrane surface appears to be functionally meaningful and may adjust CT action against different types of cells.

9. Tuuf J., Kjellberg M.A., Molotkovsky J.G., Hanada K., Mattjus P. (2011). The intermembrane ceramide transport catalyzed by CERT is sensitive to the lipid environment. Biochim. Biophys. Acta 1808 (1), 229–35 [+]

   The in vitro activity of the ceramide transporter, CERT has been studied using a fluorescence assay. CERT is responsible for the in vivo non-vesicular trafficking of ceramide between the endoplasmic reticulum and Golgi. In this study we have examined how the membrane environment surrounding the ceramide substrate, the membrane packing density and the membrane charge, are affecting the ceramide transfer activity. To examine this we have used an anthrylvinyl-labeled ceramide analogue. We found that if ceramide is in a tightly packed environment such as in sphingomyelin or dipalmitoylphosphatidylcholine containing membranes, the CERT transfer activity is markedly reduced. Ceramide in fluid membranes on the other hand are available for CERT mediated transfer. CERT also favors membranes that contain phosphatidylinositol 4-monophospate, due to its binding capacity of the pleckstrin homology domain towards phosphatidylinositol 4-monophospate. From this study we conclude that the membrane matrix surrounding ceramide, that is ceramide miscibility, is largely affecting the transfer activity of CERT.

10. Kamlekar R.K., Gao Y., Kenoth R., Molotkovsky J.G., Prendergast F.G., Malinina L., Patel D.J., Wessels W.S., Venyaminov S.Y., Brown R.E. (2010). Human GLTP: Three distinct functions for the three tryptophans in a novel peripheral amphitropic fold. Biophys. J. 99 (8), 2626–35 [+]

    Human glycolipid transfer protein (GLTP) serves as the GLTP-fold prototype, a novel, to our knowledge, peripheral amphitropic fold and structurally unique lipid binding motif that defines the GLTP superfamily. Despite conservation of all three intrinsic Trps in vertebrate GLTPs, the Trp functional role(s) remains unclear. Herein, the issue is addressed using circular dichroism and fluorescence spectroscopy along with an atypical Trp point mutation strategy. Far-ultraviolet and near-ultraviolet circular dichroism spectroscopic analyses showed that W96F-W142Y-GLTP and W96Y-GLTP retain their native conformation and stability, whereas W85Y-W96F-GLTP is slightly altered, in agreement with relative glycolipid transfer activities of >90%, ∼85%, and ∼45%, respectively. In silico three-dimensional modeling and acrylamide quenching of Trp fluorescence supported a nativelike folding conformation. With the Trp⁹⁶-less mutants, changes in emission intensity, wavelength maximum, lifetime, and time-resolved anisotropy decay induced by phosphoglyceride membranes lacking or containing glycolipid and by excitation at different wavelengths along the absorption-spectrum red edge indicated differing functions for W142 and W85. The data suggest that W142 acts as a shallow-penetration anchor during docking with membrane interfaces, whereas the buried W85 indole helps maintain proper folding and possibly regulates membrane-induced transitioning to a glycolipid-acquiring conformation. The findings illustrate remarkable versatility for Trp, providing three distinct intramolecular functions in the novel amphitropic GLTP fold.

11. Boldyrev I.A., Molotkovskiĭ J.G. (2010). New 4,4-Difluoro-3a,4a-Diaza-s-Indacene (BODIPY)-Labeled Sphingolipids for Membrane Studies. Russ. J. Bioorg. Chem. 36 (4), 508–511 [+]

    The synthesis of a series of new fluorescently labeled sphingolipids containing a 4,4-difluoro-
    1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-yl (Me4-BODIPY-8) group at the ω-position of a
    fatty acyl residue is described. The obtained probes were used in studies of biological and model membrane
    systems.

12. Sachl R., Boldyrev I., Johansson L.B.A. (2010). Localisation of BODIPY-labelled phosphatidylcholines in lipid bilayers. Phys. Chem. Chem. Phys. 12, 6027–6034 [+]

    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 C_n-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.

13. 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.

14. 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.

15. Boldyrev I.A., Gaenko G.P., Moiseeva E.V., Deligeorgiev T., Kaloianova S., Lesev N., Vasilev A., Molotkovskiĭ Iu.G. (2009). [1,10-phenantroline europium complexes: their inclusion in liposomes and cytotoxicity]. Bioorg. Khim. 37 (3), 408–13 [+]

    For a series of 1,10-phenantroline tris-beta-diketonate europium complexes (EuC), cytotoxic activity on the HBL-100 human breast carcinoma cells was determined. Liposomal preparation of the most active EuC, V12, was also tested for cytotoxicity. Testing of this preparation in vivo on starting lethal murine model of T cell leukemic lymphoma ASF-LL showed that the inclusion of V12 in liposomes did not increase its antitumour activity in a local mode of administration.

16. 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.

17. 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.

18. 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.

19. 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.

20. 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.

21. 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 mmol
    1) 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.

22. 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.

23. 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.

24. 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.

25. 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
26. 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.

27. 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

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