Jul. G. Molotkovsky

[A synthesis and properties of new 4,4-difluoro-3a,4a-diaza-s-indacene (BODIPY))-labeled lipids].

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 ω-position were synthesized. These acids were used to prepare new fluorescently labeled phosphatidylcholines, sphingomyelin, and galactosyl ceramide. The symmetry of the Me4-BODIPY-8-fluorophore suggests that, in most bilayer membrane systems, this fluorophore would be embedded into the bilayer.

Synthesis of a lipid derivative of the antitumor agent methotrexate

A lipophilic methotrexate prodrug capable of incorporation into membranes of carrier liposomes was synthesized. The conjugate consists of a lipophilic rac-1,2-dioleoylglycerol anchor connected to methotrexate through a βAla–N-carbonylmethylene linker, which should be located in the polar region of the lipid bilayer. The ester bond between the hydrophilic linker and the antitumor agent can be hydrolyzed by intracellular esterases. The liposomal formulation of the prodrug exhibited a cytotoxic activity in vitro.

Synthesis and Characteristics of Fluorescent BODIPY-Labeled Gangliosides

A series of new fluorescent-labeled gangliosides bearing the residues of acids labeled by 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene(BODIPY) in the polar or/and apolar moiety were synthesized. These are ganglioside GM1 labeled with the residue of 4,4-difluoro-4-bora-3a,4a-diaza-5,7-dimethyl-s-indacenyl-3-propanoic (BODIPY-FL-propanoic) and -indacenyl-5-pentanoic (BODIPY-FL-pentanoic) acid in the oligosaccharide moiety of the molecule, and ganglioside GD1a labeled with two residues of BODIPY-FL-pentanoic acid in the oligosaccharide moiety and also with the residue of BODIPY-FL-pentanoic acid and the residue of 4,4-difluoro-4-bora-3a,4a-diaza-5-octyl-s-indacenyl-5-pentanoic acid in the ceramide part of the molecule. Some spectral characteristics and the behavior in the model membrane systems of the synthesized probes were studied. In their emission spectra, the BODIPY-labeled gangliosides included into phosphatidylcholine liposomes at high concentrations (>1 mol %) exhibit a long-wavelength maximum (at ∼630 nm) in addition to the usual maximum (at 510–515 nm).

Synthesis and characteristics of new fluorescent probes based on cardiolipin

New fluorescent lipid probes, cardiolipin derivatives AV12-CL and B7-CL, bearing the residues of 12-(9-anthryl)-11E-dodecenoic and 7-(4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacen-8-yl)heptanoic acid, respectively, have been synthesized by acylation of 1-lysocardiolipin, which had been obtained from bovine heart cardiolipin by enzymatic hydrolysis with bacterial lipase. The resulting probes are intended for the study of protein-anionic phospholipid interactions.

Liposomal formulation of a methotrexate diglyceride conjugate: Activity toward a culture of methotrexate-resistant leukemia cells

We have recently synthesized a lipid conjugate of the anticancer agent methotrexate (MTX-DG) and showed that the conjugate is quantitatively included in the lipid bilayer of liposomes prepared by a standard extrusion technique from an 8:1:1 (mol) egg phosphatidylcholine-yeast phosphatidylinositol-MTX-DG mixture. Both the size of liposomes (126 ± 30 nm) and the MTX-DG concentration (4.4 mM) are relevant for systemic injections in mammals. The liposomal formulation of MTX-DG was shown to overcome the resistance of tumor cells in vitro to methotrexate: the cytotoxic activities (IC50) of MTX in cultures of the human T-lymphoblastic leukemia cell line CEM-CCRF and the MTX-resistant subline CEM/MTX were 0.075±0.005 and 16.4±4.9 μM, respectively, while, in the case of liposomes loaded with MTX-DG, the IC50 values were much closer: 0.77±0.06 and 3.8±1.9 μM.

[Depth-dependent investigation of the apolar zone of lipid membranes using a series of fluorescent probes, Me4-BODIPY-8-labeled phosphatidylcholines].

A series of lipid probes, phosphatidylcholines labeled with Me4-BODIPY-8 (4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacen-8-yl) fluorophore attached to the end of acyl residue at different distances from the polar head, were used as depth-dependent probes for the apolar zone of the model membrane systems, large unilamellar vesicles (LUV). Data on the anisotropy of probe fluorescence demonstrated a different mobility profiles for the fluorophore microenvironment in LUVs of different composition at various temperatures, which indicates a high sensitivity of these probes as tools for studying membrane systems. An interesting anomaly was observed for LUVs from dimiristoylphosphatidylcholine (DMPC) or from a DMPC-cholesterol mixture: the anisotropy of the fluorophore located near the bilayer center is larger than that of the fluorophore located further from the center; i.e., the mobility of the microenvironment is lower in the first case. This anomaly is supposed to result from the penetration of unlabeled long chain of the probes to the opposite bilayer leaflet. Such a possibility should be taken into account when constructing the fluorescent probes and interpreting the results.

The Synthesis of Photoaffinity Neoglycolipid Probes as Tools for Studying Membrane Lectins

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 and A trisaccharide, which are 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 crosslinked products after photoaffinity labeling.

[Fluorescent properties of 9-anthracenecarboxamides].

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.

Synthesis and Properties of a Fluorescent-Labeled Triglyceride Derivative of the Antitumor Drug Merphalan

A new fluorescent probe, a 3-perylenoyl derivative of the lipophilized antitumor drug merphalan (sarcolysine), was synthesized. The probe is suitable for studying intracellular traffic and metabolism of merphalan and its derivatives. The perylenoyl fluorescence is partially quenched by the merphalan chromophore, which broadens the probe potentialities.

[A synthesis of new rigid fluorescent bichromophoric probes for studying mechanisms of donor-donor energy migration].

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 β-Ala and L-Ser residues, which provide for a different nearest environment of the fluorophores. The third probe has identical β-Ala inserts.