Russian Academy of Sciences Institute of Chemical Biology and Fundamental Medicine SB RAS

Centre National de la Recherche Scientifique European Research Association “SupraChem”

ARCUS Alsace – Russia / Ukraine

First Symposium

Supramolecular Chemistry for Materials and Life Sciences

Abstract book

June 29 – July 3, 2010 Novosibirsk, Russia

Russian Academy of Sciences Institute of Chemical Biology and Fundamental Medicine SB RAS

Centre National de la Recherche Scientifique European Research Association “SupraChem”

ARCUS Alsace – Russia / Ukraine

First Symposium

Supramolecular Chemistry for Materials and Life Sciences

Abstract book

June 29 – July 3, 2010

Novosibirsk, Russia

International Organizing Committee Valentin Vlassov (Russia) – Chairman Galina Karpova (Russia)– Vice-chairwoman Alain Krol (France) – Vice-chairman Vladimir Fedin (Russia) Richard Giegé (France) Mir Wais Hosseini (France) Ivan Huc (France) Vitaly Kalchenko (Ukraine) Olga Lavrik (Russia) Alexandre Varnek (France) Marina Zenkova (Russia)

Local Organizing Committee

Galina Karpova, Chairwoman Dmitri Graifer, Scientific secretary Anton Ivanov Olga Klimchuk Ekaterina Kovalenko Elena Kuligina Alexey Malygin Svetlana Mysina Ekaterina Pinaeva Vladimir Richter

Organizing committee would like to thank the following for financial support:

Ruschembio, LTD www.ruschembio.ru

Helicon Company www.helicon.ёru

Bio-Rad Laboratories, LTD

www.bio-rad.com

Carl Zeiss, LTD www.zeiss.ru

Biogen-Analytika, LTD www.bga.su

Promix, LTD www.promix.ru

Leica Microsystems www.leica-microsystems.com

Oral presentations

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RUSCHEMBIO - Infrastructural Project of RUSNANO Corporation. Our aim is to build a system of delivering reagents and consumables for research institutes, quality control lab and manufactures. Key elements of the system will be a wide range of high quality goods in the stock, effective delivery of products in the territory of the Russian Federation and work with licensed and require special temperature conditions reagents. Today, the warehouses of our company have more than 10000 reagents and consumables, and at the end of 2010 the number of products will be 2 times more. The main advantage of

RUSNANO Corporation is a wide range of goods in the warehouse, ordering products through the web site, delivery of stock items within 24 hours in Moscow and 72 hours of the regions of Russia.

9

EPR study of supramolecular complexes of functional nitroxides and nanocapsules

Bagryanskaya E.1, Polovyanenko D.1, Semenov S.1, Kirilyuk I.2, Gerasko O.3,

Fedin V.3 and Khramtsov V.4 1 International Tomography Center SB RAS, Novosibirsk, 630090, Russia

2Novosibirsk Institute of Organic Chemistry SB RAS, Novosibirsk, 630090, Russia 3 Institute of Inorganic Chemistry SBRAS, Novosibirsk, 630090, Russia

4 Dorothy M. Davis Heart & Lung Research Institute, The Ohio State University, Columbus, OH 43210, USA

Encapsulation of stable free radicals in molecular nanocontainers can enhance

stability of the former against enzymes and, hence, has a good potential for application

in EPR tomography and oxidative stress studies. This report concerns application of

multifrequency EPR and NMR to study supramolecular complexes of stable nitroxides

and nanocapsules (cucurbityriles, calixarenes, cyclodexrines and polysomes)[1-7]. It is

shown that encapsulation of functional nitroxides in nano-sized containers can enhance

their persistence to reduction and improve their functional properties. Complexation

constants for the several nitroxides with cucurbityriles were obtained by EPR at various

pH and found to be pH dependent []. The effect of alkali cations on complexation of

nitroxides was studied. Using EPR we measured reduction kinetics of nitroxides by

sodium ascorbate and found significant protection of the encapsulated nitroxides against

reduction by ascorbate. These complexes can be used as more stable pH-probes for

biological applications.

The influence of methyl- -cyclodextrin on the life-time of spin adduct of three

different spin traps (PBN, DMPO and DPMPO) with glythatyil radical has been studied

using EPR [5]. It is shown that cyclodextrin plays an important role in stabilizing of the

correspondent spin adducts.

This work was supported by RFBR grant 08-04-0055 and Russian Federal

Agency for Education project N 1144.

References

1. G.S. Ananchenko, et al, Chem. Comm. (2008) 223–225. 2. D. N.Polovyanenko et al., PCCP, 10(2008)5299. 3. E.G. Bagryanskaya et al. PCCP 11 (2009),6700-6708. 4. E. G. Bagryanskaya et al., Appl. Mag. Res. 36 (2009) 181-194. 5. D.N. Polovyanenko et al., J. Phys. Chem. B.112 (2008)13157. 6. I. Kirilyuk,et al.J.Phys. Chem. B. 114 (2010) 1719-1728. 7. Y.Y. Woldman, Y. Y., Analyst.134 (2009) 904-10.

10

New calixarenes based on IL plateform Ouadi A.1, Miroshnichenko S.2, Kalchenko V.2, Billard I.1

1IPHC, DRS/CHNU, 23 rue du loess, 67037 Strasbourg Cedex 2, France 2Institute of Organic Chemistry, National Academy of Sciences of Ukraine

02660, Kiev-94, Ukraine

Ionic liquids (ILs) are compounds with a wide array of potential applications. Among

others, their use in liquid/liquid extraction is the subject of numerous studies, owing to their

interesting properties. Although most authors will put forward their “green aspects”, as ILs

are non volatile and non flammable, we think their unusual solvating properties (i.e. as

compared to traditional solvents) are even more valuable. We present extraction data for

Am3+ and Eu3+ of two different kind of “Task-Specific” ILs on which we have grafted

calixarene motives.

In one case, the calixarene part, bearing a phosphoryl unit, is grafted onto an

imidazolium structure, so that the complexing moiety is part of the cationic entity of the IL. In

this case, the counter-anion is (CF3SO2)2N- and the resulting compound is liquid at room T. In

the other case, the phosphoryl unit on the calixarene skeleton is deprotonated and therefore

acts as the anionic part of the IL, the cation being the traditional imidazolium unit. The

resulting compound is a solid.

The extraction results will be presented and discussed.

O

PO

R R

N

N

Me

4

O

PR

4

O

N

N

Me

Bu

11

Sequence-specific recognition of double-stranded DNA and optimization of

the twisted intercalating nucleic acids (TINA) forming triple helix with the

polypurine tract of the proviral HIV DNA. Boutorine A. S.1, Doluca O.2 and Filichev V. V.2

1 Muséum National d'Histoire Naturelle, RDDM, B.P. 26, 57 rue Cuvier, F-75231 Paris Cedex 05; INSERM, U565, Paris; CNRS, UMR 7196, Paris

2 Institute of Fundamental Sciences, Massey University, Private Bag 11-222, 4442 Palmerston North, New Zealand

Two synthetic compounds can form sequence-specific complexes with double-

stranded DNA: triplex-forming oligonucleotides (TFO) and polyamide minor groove binders

(MGB). TFO are highly sequence-specific but need acid pH and long polypurine tracts in the

target sequence. MGB and especially dimeric head-to-head bis-MGB recognize shorter DNA

sequences but form stronger complexes. Their covalent conjugates could be a good

compromise for both specificity and complex stability. We synthesized such conjugates, but

in the case of non-modified oligonucleotides their target affinity and specificity was

determined by only MGB component.

In order to validate oligonucleotide part of the conjugates, we used modified Twisted

intercalating nucleic acids (TINA). TINA oligonucleotides contain insertions of (R)-1-O-[4-

(1-pyrenylethynyl)phenylmethyl]glycerol residues in their sequences. Due to TINA

intercalation, they form stable triplexes with polypurine tracts of double-stranded DNA. Their

affinity depends on the oligonucleotide length, primary structure and base contents, parallel or

antiparallel orientation of oligonucleotides respectively to DNA, quantity of TINA residues

and their relative position. Basing on parallel CT, GT and antiparallel GT triplex-forming 16-

mer oligonucleotides targeted to polypurine tract of HIV proviral DNA, we synthesized 14

different oligonucleotide structures with 2-4 TINA insertions (x) in different positions.

Studies of their interaction with target duplex by gel shift, fluorescence spectroscopy, circular

dichroism and thermal denaturation permitted to compare their binding properties. In general,

antiparallel GT oligonucleotides are better than parallel TC or TG ones. Two candidates were

retained on the base of their high affinity (Kd = 0.245 and 0.040 µm, respectively). The

second one (5'-AGGGxGGGTTTxTGTTTT-3') contained 2 TINA insertions and did not

aggregate in non-denaturing conditions, in contrast to majority of other structures. The rules

for synthesis of TINA sequences forming stable triplexes with the target sequence are

presented.

12

From the first structural link between Nanosized molybdenum oxide-based

ions and derived Keggin structure to the Giant Sulfurated-Keplerate Anion Leclerc N.1, Floqueta S., Simonnet-Jegat C.1, Korenev V.2 and Cadot E.1 1ILV-UMR 8180, Universite de Versailles Saint Quentin, Versailles (France) 2 Nikolaiev Institute of Inorganic Chemistry SB RAS, Novosibirsk (Russia)

The peculiar reactivity of the monovacant ion [HBW11O39]9- was previously reported by

Teze et al. who demonstrate that addition of tungstate on [HBW11O39]8- under acidic conditions

does not lead directly to the thermodynamically stable -[BW12O40]5- saturated Keggin ion but

gives first, the unconventional Keggin derivative [H3BW13O46]8-

anion, which reveals an unusual arrangement consisting of an outer

{W3O7} core grafted on the monovacant [BW11O39]9- Keggin moiety.

[1,2] Furthermore, the {BW13} unit behaves as a lacunary

polyoxotungstate and can be used to produce mixed-metal

arrangements. Addition of molybdate on [HBW11O39]8- ion leads to

the formation of mixed pentagonal units {W(Mo5)} and {W(WMo4)}

trapped as linkers in the resulting modular assemblies, thus

establishing the first link between the Keggin ions derivatives and the

giant molybdenum oxide ions (see Figure a and b).[3-4] In the same

way, the {Mo2O2S2}2+ cations can be engaged within

polycondensation processes of oxo molybdate ions to lead to the first

sulfurated giant anions built on connections between the pentagonal

{Mo(Mo)5}units and the {Mo2O2S2} linkers. Three new compounds,

namely {Mo40S8}, {Mo63S12}, and {Mo132S60} (see Figure c) will be

presented in relationship with their amazing potentialities in the fields

of supramolecular chemistry and electrocatalysis (reduction of protons

into hydrogen).[4]

1. A. Teze, M. Michelon, G. Herve, Inorg. Chem. 1997, 36, 505-509. 2. N. Laronze-Leclerc, J. Marrot, G. Herve, R. Thouvenot, E. Cadot, Chem. Eur. J. 2007, 13 7234-7245. 3. N. Leclerc-Laronze, J. Marrot, R. Thouvenot* and E. Cadot, Angew. Chem. Int. Ed. 2008, 131, 17254. 4. Keita, B. ; Floquet, S. ; Lemonnier, J.-F. ; Cadot, E. ; Kachmar, A. ; Benard, M. ; Rohmer, M.-M. ; Nadjo, L. J. Phys. Chem. C 2008, 112, 1109.

13

Design and properties of shRNA expressing cassettes

Kuchnov D.S., Pyshny D.V., Zenkova M.A., Vlassov V.V.,

Chernolovskaya E.L.

Institute of Chemical Biology and Fundamental Medicine SB RAS, Lavrentiev ave., 8,

Novosibirsk 630090, Russia. E-mail: Elena_ch@niboch.nsc.ru

RNA interference is an evolutionary conserved mechanism of specific gene silencing induced

by double stranded RNA homologous to the target mRNA. Currently, there are two strategies

for realization of RNAi in mammalian cells: (i) design and delivery of short siRNA into cells,

and (ii) development of siRNA expression vectors and each strategy has its advantages and

problems. siRNA used in the first one have a short live time in the presence of serum and

cellular nucleases, but their properties can be improved by chemical modifications. Plasmid,

viral vectors and shRNA expressing cassettes presented by DNA fragments are more stable in

culture media, cells and organisms, and are able to express high level of shRNA for prolonged

period of time. Ultimate success of all the approaches described above depends strongly on

the sufficient cell penetration of the nucleic acids involved. The conjugation of siRNA to the

molecules, which can be internalized into the cell by natural transport mechanisms, can be a

promising approach for the delivery of shRNA expressing cassettes into the cells. Chemically

modified cassettes were prepared by PCR using primers conjugated with lipophilic molecules

via different linkers. The other approach is based on the formation of supramolecular

complexes containing shRNA expressing cassettes and oligonucleotides conjugated with the

transport molecules. shRNA expressing cassettes with specific structure efficiently penetrate

into the cells. New technology for regulation of gene expression based on shRNA gives the

opportunity to develop the therapeutics for the pharmacological control of disease-related

genes.

This work was supported by RAS programs “Molecular and Cellular Biology” and “Basic

sciences for medicine”, grant from SB RAS No. 41, RFBR No. 08-04-01073-а, 09-04-12128-

ofi_m.

14

Spectroscopy and Photophysics of ruthenium (II) polypyridyl

complexes used as DNA intercalators: a theoretical study

Ambrosek D.a, Loos P. –F.b, Assfeld X.b, Daniel C.a

a Institut de Chimie UMR 7177 CNRS/ Universitй de Strasbourg, Laboratoire de Chimie Quantique, ,4 Rue Blaise Pascal, B. P. 1032 67 070 Strasbourg Cedex France

b Laboratoire de Chimie et Biochimie Thйorique UMR 7565 CNRS / Universitй Henri Poincarй 54 506 Vandoeuvre Les Nancy France

The electronic absorption spectroscopy and the photophysics of the low-lying excited

states of [Ru(phen)2dppz]2+ and [Ru(tap)2dppz]2+ (phen = 1,10-phenanthroline; tap = 1,4,5,8-

tetraazaphenanthrene; dppz = dipyridophenazine) in various media (water, acetonitrile, bases

pairs) were investigated by means of density functional theory. The aim of the theoretical

study is to understand and to rationalize the different photophysical behaviours of the two

complexes when intercalated in DNA, namely a luminescence process, highly efficient in the

case of the phen substituted molecule, process which is quenched by fast or ultra-fast electron

transfer from the guanine to the Ru(II) complex in the case of [Ru(tap)2dppz]2+. 1-3 It is

shown that the character of the lowest 3MLCT states (dRu → *phen, dRu → *tap, dRu →

*dppz) and their position with respect to the 3IL (dppz → *dppz) state will control the

photophysics of these molecules in various media or when intercalated in polynucleotides.

Whereas the main features of the electronic absorption spectra depend strongly on the solvent

corrections and on the mode of intercalation in DNA (minor or major groove) they have little

influence on the observed processes, namely luminescence or electron transfer. 4

1. Friedman, A. E.; Chambron, J. C.; Sauvage, J. P.; Turro, N. J.; Barton J. K. J. Am.

Chem. Soc. 1990, 112, 4969; Hiort, C. H.; Lincoln, P.; Norden; B. J. Am. Chem. Soc.

1993, 115, 3448.

2. Olson, E. J. C.; Hu, D.; Hoermann, A.; Jonkman, A. M.; Arkin, M. R.; Stemp, E. D.

A.; Barton, J. K.; Barbara, P. F. J. Am. Chem. Soc. 1997, 119, 11458; Coates, C. G.;

Olofsson, J.; Coletti, M.; McGarvey, J. J.; Onfelt, B.; Lincoln, P.; Norden, B.; Tuite,

E.; Matousek, P.; Parker, A. W. J. Phys. Chem. 2001, 105, 12653.

3. Ortmans, I.; Elias, B.; Kelly, J. M.; Moucheron, C.; Kirsch-DeMesmaeker, A. Dalton

Trans 2004, 668-676.

4. Atsumi, M.; Gonzбlez, L.; Daniel, C. J. of Photochem. & Photobio A: Chem. 2007,

190, 310; Ambrosek, D.; Loos, P.-F.; Assfeld, X.; Daniel, C. J. of Inorganic

Biochemistry, accepted for publication march 2010.

15

Mechanisms involved in formation and stability of DNA repair complexes

Dianov G.

Gray Institute for Radiation Oncology and Biology Unit, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ, UK

Exogenous and endogenous mutagens induce a variety of DNA lesions including base

damages and single- and double-strand breaks which, if unrepaired, may cause human

diseases such as cancer. Although biochemical properties of DNA repair enzymes involved

in processing of DNA lesions are well characterised, the mechanisms involved in DNA

damage recognition and formation of DNA damage-specific protein complexes is unclear.

We designed a novel protocol to reveal the engagement of specific proteins during repair of

DNA single-strand breaks. This protocol employs formaldehyde crosslinking of proteins

involved in DNA repair to oligonucleotide duplexes containing site-specific DNA damage.

Using formaldehyde crosslinking during repair of DNA substrates containing different DNA

lesions we found that the formation of the DNA repair complexes is initiated by damage-

specific proteins and that repair complexes are stabilised on damaged DNA by scaffold

proteins.

16

Telomerase activity regulation

Zvereva M., Scherbakova D., Logvina N., Smekalova E., Shubernetskaya O.,

Adgibeck D., Skvotsov D., Rubtsova M., Dontsova O.

Department of Chemistry and A.N. Belozersky Institute, M.V. Lomonosov Moscow State University, Moscow, Russia.

Telomerase is an RNA-protein complex that plays a key role in telomere length

maintenance in the vast majority of eucaryotic organisms. Telomerase core consists of

catalytic reverse transcriptase protein subunit and telomerase RNA. Yeast telomerase is

known to form functional dimers in vivo, the data that it can function as a monomere in vitro

will be presented. Number of additional proteins regulates telomerase activity in vivo. The

functional properties of telomerase accessory protein Est3 and its role in the regulation of

telomerase activity and dimerization status will be discussed. The data on the attempts to

reconstitute telomerase complex in vitro will be described. The data concerning the influence

of G-quadruplexes of the basis of DNA and RNA oligonucleotides on telomerase activity

both in yeast and humans in vitro will be presented.

17

Mitochondrial diseases: modeling anti-genomic therapy by imported oligonucleotides

Entelis N.1, Comte C.1, Heckel A.-M.1, Smirnov A.1,2, Pyshnyi D.3, Meschaninova M.3, Venyaminova A.3, Martin R. P.1, Tarassov I.1

1 UMR 7156 UdS/CNRS, 21 rue René Descartes 67084, Strasbourg, France 2 Department of Molecular Biology, Moscow State University, Moscow 119899, Russia

3 Inst. of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk 630090, Russia Mitochondria are essential organelles of eukaryotic cells, taking part in several critical

cellular processes. They contain their own genome (mtDNA) packaged into supramolecular nucleoprotein complexes. Mutations in mtDNA have been associated with a wide variety of human disorders. In the patients with mtDNA defects, it is common to find mutant and normal (wild type) mtDNA molecules in the same cell, a situation known as heteroplasmy. Manifestation of biochemical and clinical defects occurs only when a threshold level of heteroplasmy (>60%) has been reached. Since there is no effective treatment for these disorders, one attractive approach would be to specifically target mutant mtDNA to prevent it from replicating, thereby allowing propagation of only wild-type genomes.

The limitations of this strategy consist in 2 problems: translocation of the anti-genomic oligomers through the double mitochondrial membrane, and their access and specific binding to mutated region of mtDNA. Our study of the natural pathway of RNA import into yeast and human mitochondria permitted to identify the import determinants in tRNA and 5S rRNA structures. Basing on these data, a set of small RNA molecules with significantly improved efficiency of import into yeast and human mitochondria was constructed (1). To create a vector system able to target therapeutic oligonucleotides into deficient human mitochondria, we inserted into these RNAs short sequences corresponding to the boundaries of a large deletion in mtDNA associated with a neuromuscular syndrome KSS. Recombinant RNAs, introduced into cultured human cells containing KSS deletion, were shown to be stable in the cytosol, partially imported into mitochondria, and induced a transient decrease of the mutant mtDNA proportion, thus validating the potential of our approach to rescue the deleterious mtDNA mutations. To stabilize the effect of the anti-genomic RNAs, the oligonucleotides containing chemical modifications as well as RNA/DNA chimeras were synthesized, inserted into vector RNAs and tested. To improve the anti-replicative capacity of our constructions, the selectivity and stability of their binding to mutant mtDNA should be increased using different chemical approaches. (1) Smirnov et al. (2008) RNA 14, 749; Kolesnikova et al. (2010) RNA 16, in press. This work was supported by grants AFM, ANR, FRM, RFBR and ARCUS.

18

Homochiral metal-organic coordination polymers and related studies

Fedin V. P.

Nikolaev Institute of Inorganic Chemistry SB RAS, 3 Lavrentiev av., Novosibirsk 630090, RUSSIA, Email: cluster@niic.nsc.ru

Enantiopure (homochiral) porous absorbents provide great opportunities for

stereoselective sorption of chiral guest molecules and therefore highly demanded for

separation and purification of important substrates including drugs and other bioactive

molecules. Porous homochiral coordination polymers represent promising class of porous

chiral absorbents due to high versatility of structural design, however the synthetic

accessibility still remains a challenging problem here. Recently we introduced a new synthetic

approach toward porous homochiral coordination polymers, which allows us to design series

of isotypical homochiral frameworks with similar structure of chiral centers and tunable pore

size. Starting from enantiopure (+)-camphoric acid (H2camph) we obtain a series of

homochiral porous coordination polymers with isoreticular topology [M2camph2L] (M = Zn2+,

Cu2+; L = diazabicyclo[2.2.2]octane, 4,4-bipyridil, trans-bis(4-pyridil)ethylene). These

structures share the same building chiral motif with rigid linkers (L) controlling the important

structural properties (the pore sizes, free accessible volumes) and stability of the metal-

organic frameworks upon guest exchange.

Another family of homochiral porous coordination polymers was built from enantiopure

lactic or mandelic acids [Zn2(xdc)(L*)], where xdc = bdc (terephthalate), ndc

(2,6-naphthalenedicarboxylate) or bpdc (4,4 -biphenyldicarboxylate); L* = lactate,

mandelate). These structures were shown to possess remarkable size- and enantioselective

sorption properties toward chiral alcohols and sulfoxides with enantiomeric excess up to 60%.

More important, some chiral drug molecules also show notable enantioselectivity upon

inclusion into these homochiral porous coordination polymers. The ab initio calculations of

host-guest interactions not only fully support experimental data, but provide important

insights into the nature of enantioselectivity and further developments of chiral porous

absorbents for the fine drug purification.

A grant of the Russian Academy of Sciences (program No. 5.6.1) and a grant of the

Siberian Branch of the Russian Academy of Sciences (program No. 107) are gratefully

acknowledged.

19

Coordinated conformational changes of enzymes and DNA accompany

lesion recognition and catalysis in base excision repair pathway Fedorova O. S.

Institute of Chemical Biology and Fundamental Medicine, Siberian Division of Russian Academy of Sciences, 630090 Novosibirsk, Russia, E-mail: fedorova@niboch.nsc.ru

DNA bases of all organisms are readily oxidized and alkylated in vivo. The resulting

lesions are usually repaired by base excision repair (BER) pathway. In human cells BER

pathway repairs 10,000 lesions per cell per day. The key enzymes in BER are DNA

glycosylases, which recognize a variety of modified or mismatched bases and catalyze

cleavage of the N-glycosidic bond to release the inappropriate base from the deoxyribose ring.

Many glycosylases also catalyze a -elimination (or lyase) reaction to effect strand scission

after the base removal. Subsequent action of apurinic-apyrimidinic (AP) endonucleases and

3’-phosphodiesterases remove the remaining sugar fragment to produce a single-nucleotide

gap with the proper 3’-OH and 5’-phosphate termini, a substrate for DNA polymerases. After

the DNA polymerase adds the correct nucleotide, DNA ligase completes the BER process.

Molecules of bacterial Fpg and eukaryotic OGG1 do not have sequence homology or

similar structures. In spite of this, they both are able to remove 8-oxoguanine, an abundant

pre-mutagenic oxidized nucleobase, from DNA. Recently we have investigated the

conformational transitions in several DNA repair enzymes, including DNA glycosylases (E.

coli Fpg and Nei, human OGG1) and AP endonucleases (human APE1), and in their DNA

substrates by stopped-flow detection of tryptophan and 2-aminopurine fluorescence as well as

using FRET labels in DNA. DNA substrates contained damaged bases or abasic sites of

different natures. In all cases, multiple transient changes in fluorescence intensities of

enzymes and DNA substrates were observed, indicating sequential conformational changes in

both macromolecules during the catalytic cycle. We have also applied MS/ESI to follow

appearance and disappearance of transient covalent intermediates between DNA-glycosylases

(Fpg and hOgg1) and the substrate DNA. Together with kinetic analysis of fluorescence

traces these data provide new insight into the mechanisms for DNA lesion recognition and

conversion.

The research was supported by grants from RFBR (No 08-04-12211, 10-04-00070), SB RAS

(No 28, 48), Russian Ministry of Education and Science (№ 02.740.11.0079, №

02.740.11.5012 and NS 3185.2010.4).

21

CCoooorrddiinnaattiioonn aanndd NNeettwwoorrkkss bbaasseedd oonn TThhiiaaCCAAlliixx[[44]]aarreennee

Ferlay S.a, Ovsyannikov A.a, Kozlova M.a,b, Gehin A.a ,Hosseini M.W.a, Solovieva S.E.b,

Antipin I.S.b

a Laboratoire de Chimie de Coordination Organique, UMR CNRS 7140, Université de

Strasbourg, Institut Le Bel, 4, rue Blaise Pascal, F-67000 Strasbourg, France. b A.E. Arbuzov Institute of Organic and Physical Chemistry, Russian Academy of Science,

Arbuzov str. 8, Kazan 420088, Russian Federation and Kazan State University, Kremlevskaya str. 18, Kazan 420008, Russian Federation

The macrocyclic thiacalixarene (TCA, figure 1), where X = O or S, and R = t-Bu or H,

presents some coordinating properties towards transition metals, and new interesting metallic

clusters [1] are presented, together with their physical properties.

S

*

R

*

XH 4 Figure 1

TCA has been modifed in order to obtain new divergent ligands that allow the possiblities to

form molecular networks. Some examples of new TCA ligands are presented in figure 2 (X =

O or S, and R = t-Bu or H, and n =1 or 3), together with their extended metallic network in the

crystalline state [2].

S

*

R

*

X

N 4

n

S

*

R

*

X

4N

S

*

R

*

X

4

n

N Figure 2

[1] Y.Bi, X.T.Wang, W.Liao, X.Wang, , X.Wang, H.Zhang, J.Am.Chem.Soc, 2009, 131, 11650. [2] (a) M.N. Kozlova, S. Ferlay, S.E. Solovieva, I.S. Antipin, A.I. Konovalov, N. Kyritsakas, M.W. Hosseini, Dalton Trans., 2007, 5126 ; (b) M.N. Kozlova, S. Ferlay, S.E. Solovieva, I.S. Antipin, A.I. Konovalov, N. Kyritsakas, M.W. Hosseini, Chem. Commun. , 2009, 2514.

22

Supramolecular chemistry and biology of tRNA and aminoacyl-tRNA

synthetases Richard Giegé

Architecture et Réactivité de l'ARN, Université de Strasbourg, CNRS, IBMC, 15 rue René Descartes, 67084 Strasbourg cedex, France.

Transfer RNAs (tRNA) and aminoacyl-tRNA synthetases (aaRS) are ancient

molecules present in all domains of life. Beside defining the 'second genetic code' that

governs correct expression of the genetic code at the translational level, they participate in a

variety of other cellular functions. Understanding their biology changed when massive

sequencing and high throughput structural biology combined with advanced molecular

biology entered the game and produced a wealth of novel data. The processing of the

molecular information stored in tRNA and aaRS structures, i.e. required for specificity of

tRNA aminoacylation by aaRSs, will be discussed. Understanding the physical-chemistry of

such processing remains elusive, especially when functioning in cellular environments is

concerned. The effect of subtle structural features (such as the presence of modified

nucleosides in tRNA and phylum or species specific subdomains in aaRSs) that tune tRNA

and aaRS activity will be discussed in the light of evolution. These various facets provide a

robust rational underlying the tRNA/aaRS world and converge towards its supramolecular

understanding. It is anticipated that the emerging novel knowledge will help better

understanding tRNA and aaRS dysfunctions and thus the correlated human diseases.

Five selected references: (i) Giegé R., Sissler M. & Florentz C. (1998) Universal rules and

idiosyncratic features in tRNA identity. Nucleic Acids Res. 26, 5017–35; (ii) Ryckelynck M.,

Masquida B., Giegé R. & Frugier M. (2005) An intricate RNA structure with two tRNA-

derived motives directs complex formation between yeast AspRS and its mRNA. J. Mol. Biol.

354, 614–29; (iii) Giegé R. (2008) Toward a more complete view of tRNA biology. Nature

Struct. Mol. Biol. 15, 1007–14; (iv) Giegé R., Touzé E., Lorber B., Théobald-Dietrich A. &

Sauter C. (2008) Crystallogenesis trends of free and liganded aaRSs. Crystal Growth &

Design 8, 4297–306; (v) Pütz J., Giegé R. & Florentz C. (2010) Diversity and similarity in the

tRNA world: overall view and case study on malaria-related tRNAs. FEBS Lett. 584, 350–8.

23

Biomimetic Molecular Recognition with Cross-Linked Hydrophilic Polymer

and Macrocyclic Receptors

Gorbatchuk V.V., Ziganshin M.A.

A.M. Butlerov Chemical Institute, Kazan State University, Kremlevskaya St. 18, 420008, Kazan, Russia

Key criteria of biomimetic molecular recognition were elaborated in the studies of the

binding properties of dried and hydrated proteins, hydrophilic polymers, beta-cyclodextrin,

dendrimers, dipeptides and guest-free solid calixarenes for volatile substrates in binary and

ternary systems. These criteria include the cooperative hydration effect increasing the receptor

affinity and selectivity for hydrophobic compounds [1]. The related cooperative effect is a

two-step pseudopolymorphic transition observed for the studied tert-butylthiacalix[4]arene

derivative, which enables a single sensor detection of component in a vapor mixture.

The binding properties of the studied receptors and the products of their saturation with

substrate vapors were studied using gas chromatographic headspace analysis, simultaneous

thermogravimetry and differential scanning calorimetry combined with mass-spectrometry of

evolved gases and vapors, quartz microbalance sensors, atomic force microscopy and X-ray

powder diffraction method. For systems with guest vapors and solid receptor, the vapor

sorption isotherms, Gibbs energy of clathrate formation, stoichiometry of saturated clathrates,

parameters of guest elimination and of host polymorphic transitions were determined.

The data obtained revealed a number of other receptor properties, which may be regarded

as biomimetic. Among them is cooperative increase of water-mimic (good) solvents uptake

by cross-linked poly(acrylamide) derivative in the presence of small additives of hydrophobic

(bad) components. A dependence of polymorphic collapse parameters on the host prehistory

was found for several calixarenes. The studied dendrimers were observed to have different

binding sites for different substrates. The guest substitution in solid phase was found to be an

effective regeneration method for hydrophobic hosts [2]. The results may be used as a

guideline for design of biomimetic and biocompatible materials.

The work was supported by RFBR No.08-03-01107-а, and Russian Agency of Education

No. 2.1.1/1092.

1. Gorbatchuk, V.V.; Mironov, N.A.; Solomonov, B.N.; Habicher, W.D.,

Biomacromolecules 2004, 5, 1615 -1623

2. Yakimova, L.S.; Ziganshin, M.A.; Sidorov, V.A.; Kovalev, V.V.; Shokova, E.A.;

Tafeenko, V.A.; and Gorbatchuk, V.V., J. Phys. Chem. B 2008, 112, 15569-15575

24

EUKARYOTE-SPECIFIC FEATURE OF THE RIBOSOME SITE

WHERE mRNA CODON IS DECODED

Graifer D., Khairulina Y., Bulygin K., Ven’yaminova A. and Karpova G.

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian

Academy of Sciences, Novosibirsk, 630090

In all organisms the genetic information is decoded on ribosomes where sequences of

trinucleotide codons of mRNAs are translated into polypeptide chains of the synthesized proteins.

In the course of decoding, the anticodon of the aminoacyl-tRNA recognizes the mRNA codon at

the ribosomal acceptor (A) site that ensures incorporation of the correct aminoacyl residue into the

nascent polypeptide chain. The structure of the decoding site of the prokaryotic ribosome has been

deciphered at the atomic level by X-ray crystallographic analysis, which is not yet applicable for

studying the eukaryotic ribosome. Investigations of the decoding site of mammalian ribosome by

site-directed cross-linking applying various mRNA analogues bearing photoactivatable groups

revealed ribosomal protein S15 as a key component of the decoding site although its prokaryotic

counterpart, S19p, is located away from the mRNA binding track on the ribosome.

In the present study, we determined the oligopeptide of S15 neighboring the A site mRNA

codon on the human ribosome with the use of mRNA analogues bearing perfluorophenyl azide-

modified nucleotides in the sense or stop codon targeted to the A site. The protein was cross-

linked to mRNA analogues in specific ribosomal complexes that were obtained in the presence of

polypeptide chain release factor eRF1 with mRNAs bearing stop codon. Digestion of modified

S15 with various specific proteolytic agents followed by identification of the resulting modified

oligopeptides showed that cross-link was in decapeptide 131-PGIGATHSSR-140 in the C-terminal

fragment in all cases. The results indicate an involvement of the mentioned decapeptide in the

formation of the ribosomal decoding site during elongation and termination of translation.

Alignment of amino acid sequences of eukaryotic S15 and its prokaryotic counterpart, S19p from

eubacteria and archaea, revealed that decapeptide in positions 131-140 is conserved in eukaryotes

and archaea but has no homology with C-terminal part of eubacterial S19p, which suggests

involvement of the decapeptide in the translation process in an eukaryote-specific manner.

This study was supported by RFBR grant # 08-04-00508 to G.K. and by the Russian Academy

of Sciences Presidium program “Molecular and cell biology” (grant to G.K.).

25

Molecular Design of Light-Sensitive Supramolecular Systems Based on

Unsaturated and Macrocyclic Compounds

Gromov S.P.1, Ushakov E.N.2, Vedernikov A.I.1, Kuzmina L.G.3, Alfimov M.V.1

1 Photochemistry Center of the RAS, Moscow,

2 Institute of Problems of Chemical Physics of the RAS, Chernogolovka, Moscow; 3 N. S. Kurnakov Institute of General and Inorganic Chemistry of the RAS, Moscow

A new trend is being currently formed in nanotechnology, namely, organic

nanophotonics. We propose a new unique class of polyfunctional light-sensitive compounds:

crown-containing unsaturated dyes functioning as photochromes, fluorophores and

ionophores. A large body of research has been performed for their synthesis, determination of

their spatial structures, study of self-assembly features to give nanosized systems, and also

study of fluorescent, photochemical and complexing properties.

Resulting from the research, we elaborated for the first time universal molecular

meccano, allowing one to accomplish building-up, with using a limited number of

complementary compounds, light-sensitive and light-emissive nanosized systems of varied

architecture with adjusted properties. Within the same class of compounds one can construct

in solution, solid and at the air-water interface new types of molecular switches,

photocontrolled molecular machines, photosensitive monolayers and monocrystals susceptible

to all of the key photoprocesses.

molecular devices molecular machinescucurbituril

h

PHOTOANTENNA

displacement

h

CROWNPHOTOANTENNA

Mn+

The high practical value of these studies deserves attention. They provide a new

strategy for the design of materials for nanophotonics, which was demonstrated, first of all, by

the creation of practically important sensor and photochromic materials.

This work was supported by the Presidium and the Division of the RAS, the Ministry of

Science and Education, the RFBR, the Moscow Government, the INTAS, the CRDF and

International Science Foundation (ISF), the DFG, and the Royal Society.

26

Controlling molecular movements: molecular gates and turnstiles Hosseini M.W.

Université de Strasbourg, Institut Universitaire de France, Institut Le Bel, UMR CNRS 7140, Tectonique Moléculaire du Solide, 4, rue Blaise Pascal, 67000 Strasbourg, France,

hosseini@unistra.fr

Movement plays a fundamental role in the living world. Biological motors of the

linear type based on myosine1 or kinesine2 have been discovered and studied. A rotary motor

based on ATPase has been also described.3 A molecular motor may be defined as a molecular

architecture for which a movement may be induced by external stimuli. The induced and

controlled movement must take place between a fixed and a mobile portion. In principle, one

may envisage either translational or rotational motors.4

As a first step towards molecular motors, we have designed a molecular system based

on a porphyrin core bearing at the meso positions interactions sites as a stator (Fig. 1a),

octahedral Sn(IV) cation located at the centre of the porphyrin as a hinge (Fig. 1b) and

different handles also equipped with recognition sites connected to the porphyrin through Sn-

O axial bonds (Fig. 1c). The design, synthesis and structural characterisation, both in solution

by 1H-NMR and in the solid state by X-ray diffraction on single crystals, of a series of

molecular gates and turnstiles based on porphyrin derivatives will be presented.5-7

References 1 J. T. Finer, R. M. Simmons, J. A. Spudlich, Nature, 1994, 368, 113; M. Whittaker, E. M. Wilson-Kubalek, J. E. Smith, L. Faust, R. A. Milligan, H. L. Sweeney, Nature, 1995, 378, 748. 2 E. P. Sabin, F. J. Kull, R. Cook, R. D. Vale, R. J. Fletterick, Nature, 1996, 380, 555; E. Meuhöfer, J. Howard, Proc. Natl. Acad. Sci. U.S.A, 1995, 92, 574. 3 T. Elston, H. Wang, G. Oster, Nature 1998, 391, 510; H. Noji, R. Yasuda, M. Yoshida, K. Kinosita Jr., Nature 1997, 386, 299. 4 J.-P. Sauvage, Science, 2001, 291, 2105; J. F. Stoddart, Acc. Chem. Res, 2001, 34, 410; N. Koumura, R. W. J. Zijlstra, R. A. van Delden, N. Harada, B. L. Feringa, Nature 1999, 401, 152; E. R. Kay, D. A. Leigh, F. Zerbetto, Angew. Chem. Int. Ed, 2007, 46, 72. 5 A. Guenet, E.Graf, N.Kyritsakas, L. Allouche, M. W. Hosseini, Chem. Commun. 2007, 2935. 6 A Guenet, E. Graf, N. Kyritsakas, M. W. Hosseini, Inorg. Chem. 2010, 49, 1872. 7 T. Lang, A. Guenet, E. Graf, N. Kyritsakas, M. W. Hosseini, Chem. Commun. 2010, DOI: 10.1039/b927112k.

27

Foldamers: expanding the chemical space

Huc I.

Institut Européen de Chimie Biologie, CNRS - Université de Bordeaux UMR5248 2 rue Robert Escarpit 33607, Pessac, France), i.huc@iecb.u-bordeaux.fr

Our group has developed helical foldamers – oligomers that adopt stable helical folded

conformations – derived from aromatic amino acids.1 Some of these folded objects have

shown unprecedented conformational stability,2 and constitute convenient building blocks to

elaborate synthetic, very large (protein-sized) folded architectures (Fig. 1).3 They possess a

high propensity to assemble into double, triple and quadruple helices.4 Cavities can be

designed within such synthetic molecules that enable them to act as artificial receptors5

including for chiral guests. Water soluble analogues of these foldamers show promise in

nucleic acid recognition.6

Figure 1. Crystal structure of a large foldamer comprised of two helices of opposite handedness at a 90° angle. The protein crystal structure on the right is shown as the same scale for size comparison. References 1 S. Hecht, I. Huc (Eds), Foldamers: Structure, Properties, and Applications, 2007, Wyley-VCH,

Weinheim, ISBN-13: 978-3-527-31563-5. 2 H. Jiang, J.-M. Léger, I. Huc, J. Am. Chem. Soc. 2003, 125, 3448; N. Delsuc, T. Kawanami, J.

Lefeuvre, A. Shundo, H. Ihara, M. Takafuji, I. Huc ChemPhysChem 2008, 9, 1882. 3 C. Dolain, J.-M. Léger, N. Delsuc, H. Gornitzka, I. Huc Proc. Natl. Acad. Sci. (USA) 2005, 102,

16146; 5 N. Delsuc, J.-M. Léger, S. Massip, I. Huc Angew. Chem. Int. Ed. 2007, 46, 214; D. Sánchez-García, B. Kauffmann, T. Kawanami, H. Ihara, M. Takafuji, M.-H. Delville, I. Huc, J. Am. Chem. Soc. 2009, 131, 8642.

4 Q. Gan, C. Bao, B. Kauffmann, A. Grélard, J. Xiang, S. Liu, I. Huc, H. Jiang, Angew. Chem. Int. Ed. 2008, 47, 1715; D. Haldar, H. Jiang, J.-M. Léger, I. Huc, Angew. Chem. Int. Ed. 2006, 45, 5483; Y. Ferrand, A. Kendhale, J. Garric, B. Kauffmann, I. Huc, Angew. Chem. Int. Ed. 2010, 49, in press.

5 C. Bao, B. Kauffmann, Q. Gan, K. Srinivas, H. Jiang; I. Huc Angew. Chem. Int. Ed. 2008, 47, 4153.Einstein, A. J. Am. Chem. Soc. 1939, 45, 4532.

6 P. S. Shirude, E. R. Gillies, S. Ladame, F. Godde, K. Shin-ya, I. Huc, S. Balasubramanian, J. Am. Chem. Soc. 2007, 129, 11890.

28

Interaction of Ku antigen with abasic sites

Ilina E. S., Lavrik O. I., Khodyreva S. N.

Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk

One of the most abundant lesions in DNA is abasic (AP) sites arising spontaneously or

as intermediates in base excision repair. Residues of deoxyribose in AP sites are in

equilibrium between cyclic furanose and acyclic aldehyde forms. The presence of aldehyde

function determines the ability of AP site to form Schiff base intermediate with primary

amino groups of proteins. This intermediate can be stabilized by NaBH4 treatment and,

therefore, AP DNA can be used as an approach in searching proteins, which interact with AP

sites. In HeLa cell extract, a predominant product with an apparent molecular mass of 95 kDa

was observed. Analogous covalent adducts of proteins with AP DNA were revealed in the

extracts derived from human fibroblast, HL-60, K562, and several melanoma cell lines unlike

bovine testis nuclear extract. The cross-linked protein was identified as the p80 subunit

(Ku80) of Ku antigen (Ku) by immunoprecipitation and MALDI-TOF-MS analysis. Ku is an

abundant DNA end binding protein in human cells. Ku is the DNA binding component of

DNA-dependent protein kinase (DNA-PK). AP DNAs of different structure were used to

study peculiarities of Ku interaction with DNA. Considering the extreme selectivity of AP

site-containing DNA probes, we decided to examine the use of this approach for measuring

levels of Ku in different cell extracts. We found that the amount of Ku80 estimated by dot-

ELISA and AP DNA cross-linking were comparable. Cross-linking using AP DNA allows

revealing truncated variants of Ku80 polypeptide (Ku80v). The ability of Ku80v/Ku70

heterodimer to interact with DNA-PK catalytic subunit (DNA-PKcs) is greatly reduced and

resulted in increased sensitivity to some DNA damage agents as a consequence of reduced

DNA repair. Thus, the ability of Ku80 to form cross-link with baseless deoxyribose can be

used as an efficient and easy assay to test the content of Ku antigen in cell extracts. This

approach, unlike western blot or estimation of the Ku content based on mRNA levels, reveals

forms of Ku that are active in DNA binding, including those, which have aberrations in Ku80,

but retain ability to bind DNA. In addition this test is less sensitive to the reaction conditions

than the electrophoretic mobility shift assay.

This work was supported by RFBR, project 09-04-93106, Program of RAS “Molecular and cellular Biology”, and State contract 02.740.11.0079.

29

Photoinduced processes in fluorescent Ca2+ receptors

Batat P.1,2, Lavie-Cambot A.2, Vives G.2, McClenaghan N.D.2 and Jonusauskas G.1

1 Centre de Physique Moléculaire Optique et Hertzienne, UMR-CNRS 5798,

2 Institut des Sciences Moléculaires, UMR-CNRS 5255, Université Bordeaux 1, 351 Cours de la Libération, 33405 Talence, France,

E-mail: p.batat@cpmoh.u-bordeaux1.fr

Most of the cellular systems are using Ca2+ for regulating their intercellular functions.

To understand cellular Ca2+, one must be able to measure it1. Therefore the research on

synthetic Ca2+ receptors is important. BAPTA2 (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-

tetraacetic acid) (developed by Tsien, Nobel Prize 2008) is quite useful since in the presence

of Ca2+ , chromophore (see figure) emission may be altered spectacularly. This motif has been

exploited in the development of various fluorescent supramolecular systems by different

groups including our own3,4. In this report we will present ultrafast studies on some new Ca2+

receptors containing anthracenes, coumarins and BODIPYs as chromophores.

BAPTA – Ca2+

= Ca2+

N

O

O

O-

OO-

N

O

O

-O

O-O

Chromophore Chromophore

1. E. Carafoli, B. Klee, Calcium as a cellular regulator, Oxford University Press, 1999 2. R. Y. Tsien, Biochemistry, 19 (1980) 2396. 3. A. P. de Silva, N. D. McClenaghan, J. Am. Chem. Soc., 122 (2000) 3965. 4. A. P. de Silva, N. D. McClenaghan, Chem. Eur. J., 8 (2002) 4935.

We thank Bordeaux1 University, Région Aquitaine, ERC (FP7/2007-2013) “Ideas” program agreement N° 208702 and GDRI “Suprachem” for financial support.

30

Calixarene receptors for molecules and ions

Kalchenko V.

Institute of Organic Chemistry, National Academy of Sciences of Ukraine 02660, Kiev-94, Ukraine, vik@ioch.kiev.ua, www.ioch.kiev.ua/calix

Calixarenes are versatile molecular scaffolds for design of highly efficient and selective

receptors, self-assembling systems and well defined functional nanostructures. The paper will

report the molecular modeling, synthesis, structural investigations of phosphorus, nitrogen

and sulfur containing (thia)calixarenes and their supramolecular complexes with a series of

bio-relevant or ecologically hazardous molecules, cations and anions.

The special attention will be paid to chiral and water-soluble calixarenes in context of

bio-medical investigations. An application of the P,N,S-containing calixarenes toward

radionuclides extraction, chemosensors constructions, functional nanoparticles formations and

drug design will be discussed.

References: [1] For review see: Cherenok S., Dutasta J.-P., Kalchenko V. Current Organic Chemistry. 2006.10. 2307-2331; Kalchenko V. UPAC. 2008. 80. 1449-1458; Rodik R.V., Boyko V.I., Kalchenko V. I. Current Medicinal Chemistry. 2009. 16 (13), 1630-1655. Cherenok S., Kalchenko V. Topics in Heterocyclic Chemistry. 2009. 20. 229-273. [2] Kasyan O., Kalchenko V., Bolte V., Bohmer V. Chem. Commun. 2006. 1932–1934. [3] Cherenok S., Vovk A., Muravyova I., Shivanyuk A., Kukhar V., Lipkowski J., Kalchenko V. Organic Letters. 2006. 8. 549-551. [4] Notestein J.M., Andrini L.R., Kalchenko V.I., Requejo F.J., Katz A., Iglesia E. J. Am. Chem. Soc. 2007. 129. 1123-1131. [5] Cherenok S., Vovk A., Muravyova I., Shivanyuk A., Kukhar V., Lipkowski J., Kalchenko V.Organic Letters. 2006. 8. 549-552. [6] Torgov V.G., Us T.V., Korda T.M., Kostin G.A., Miroshnichenko S.I., Klimchuk O.V., Kalchenko V.I. J. Inclusion Phenomena and Macrocyclic Chemistry. 2008. 62. 51-58. [7] Klyachina M.А., Yesypenkо O.A., Pyrozhenko V.V., Shishkina S.V., Shishkin O.V., Boyko V.І., Kalchenko V.I. Tetrahedron. 2009. 65. 7085-7091. [8] Ha J.-M., Katz A., Drapailo A.B., Kalchenko V. I. J. Phys. Chem. C. 2009. 113. 1137-1142. [9] Arnaud-Neu F., Karavan M., Hubscher-Bruder V., Smirnov I., Kalchenko V. J. Inclusion Phenomena and Macrocyclic Chemistry. 2010. 66. No 1-2. 113-123. [10] Vovk A.I., Kononets L.A., Tanchuk V.Yu, Cherenok S.O., Drapailo A.B., Kalchenko V.I., Kukhar V.I. Bioorg. Med. Chem. Lett. 2010. 20. 483–487.

31

Stopped-flow conformational study of abasic site repair by specific AP

endonucleases from human and yeast S. cerevisiae

Kanazhevskaya L.Yu. 1,*, Dyakonova E.S.1,2, Koval V.V.1,2 and Fedorova O.S.1,2

1 Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian

Academy of Science 2 Novosibirsk State University, Novosibirsk 630090, Russia, amyp@niboch.nsc.ru

Apurinic/apyrimidinic (AP) sites belong to the most common DNA lesions arising as a

result of spontaneous degradation and the action of various metabolic and exogenous factors.

This type of DNA damage is highly mutagenic and cytotoxic, since it is non-instructive for

DNA polymerases and is easily converted to single- and double-strand breaks. To remove

such lesions living cells have different repair systems, including base excision repair

pathways (BER). The crucial enzymes of BER pathway in S. cerevisiae and human cells are

AP endonucleases (APN1 and APE1, respectively), which recognize AP sites in dsDNA and

make a single nick in the phosphodiester backbone 5' to the AP site. Correct functioning of

these enzymes is required for the genomic DNA protection and cell survival.

In present study the stopped-flow approach in combination with 2-aminopurine

fluorescence detection was employed to investigate a conformational dynamics and transient

kinetics of APE1 and APN1 enzymes in supramolecular complexes with damaged DNA.

Fluorescent analogue of adenine, 2-aminopurine (2aPu), was introduced into 12 bp DNA

duplexes containing the natural abasic site or its tetrahydrofuran analog in the middle of the

modified strand. Changes in the 2aPu fluorescence intensity are indicative of conformational

transitions in the DNA substrate molecule. Fluorescent traces, obtained under single-turnover

conditions, demonstrate a multi-stage character of the enzymatic processes studied. The

quantitative analysis of fluorescent data has shown a high rate of reactions catalyzed by these

AP endonucleases. The values of determined kinetic constants suggest that human APE1

protein cleaves specific DNA substrates approximately 8-times faster, than APN1 from yeast.

This research was supported by grants from RFBR (No 10-04-00070), SB RAS (No 28, 48, 90,

21), Russian Ministry of Education and Science (No NS-3185.2010.4), State Contracts (No

02.740.11.0079, 02.740.11.5012).

32

RNAs carrying cross-linkers at specific as tools for studying cellular protein

synthesizing bionanomachinery

Graifer D., Ven’yaminova A. and Karpova G.

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian

Academy of Sciences, Novosibirsk, 630090

In all kingdoms, from bacteria to human, proteins are synthesized by specialized cellular

organelles, ribosomes, according to the program incoming as messenger RNAs (mRNAs) whose

nucleotide sequences are copied from genomic DNA. Ribosomes are molecular machineries of

about 20 nm size composed of two subunits, each contains ribosomal RNAs and several dozens of

proteins. Their functioning is based on principles of self-assembly, self-organization, molecular

recognition and autoregulation that are typical features of complex supramolecular structures.

During protein synthesis, various specific RNA ligands bind to the ribosome, and one of the key

ligands is mRNA bearing genetic information to be translated into the sequence of amino acids of

the protein. Highly sensitive tools to investigate molecular environment of RNA ligands on the

ribosome are RNA derivatives bearing a perfluorophenyl azide cross-linker at specific locations.

Using short mRNA analogues, oligoribonucleotides derivatized at designed locations, enables to

obtain specific model complexes where position of mRNA analogue on the ribosome is exactly

fixed by tRNA cognate to the selected mRNA codon. Mild UV-irradiation of such complexes

results in cross-linking of mRNA analogue to the neighbor ribosomal components. Using this

approach, we learned the molecular architecture of mRNA binding site of human ribosomes that

could not be studied by X-ray crystallography so far, at the level of rRNA nucleotides, ribosomal

proteins and even oligopeptide fragments of proteins. Recently, we suggested a novel strategy

making possible to selective introduce cross-linkers into long structured RNAs based on site-

specific modification of RNA with reactive derivatives of deoxy-oligomers complementary to a

sequence adjacent to the target site. Application of this strategy to study binding site of IRES-

element of hepatitis C virus on the ribosome made it possible to reveal ribosomal proteins forming

this site that could be considered as potential targets for new antiviral drugs. Evidently, site-

specific modified RNAs are very suitable tools for studying architecture of any complex

ribonucleoprotein.

This study was supported by RFBR grant # 08-04-00508 to G.K. and by the Russian Academy of Sciences Presidium program “Molecular and cell biology” (grant to G.K.).

33

Conserved motif GTx in positions 31-33 of translation termination factor

eRF1 neighbors stop codon of mRNA in the human ribosome

Khairulina Y1., Bulygin K1., Graifer D1., Ven’yaminova A1., Frolova L2., Karpova G1.

1Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian

Academy of Sciences, Novosibirsk, 630090, Russia 2Engelhardt Institute of Molecular Biology, the Russian Academy of Sciences, Moscow

119991, Russia

Translation termination in eukaryotes ensures with high fidelity the formation of

normal-sized proteins, and takes place when one of the three stop codons, UAA, UAG or

UGA, is translocated to the ribosomal aminoacyl (A) site where it is recognized by

polypeptide chain release factor eRF1 that triggers hydrolysis of the ester bond between the

peptidyl and tRNA moieties of the peptidyl-tRNA bound at the peptidyl (P) site.

In this study, we have applied a cross-linking approach to obtain information on

positioning of purines of stop signal towards eRF1 in the terminating ribosome. We have used

a set of 32P-labeled photoactivatable mRNA analogues bearing perfluorophenyl azide cross-

linkers at mRNA positions +5 to +7 with respect to the first nucleotide of the codon targeted

to the P site by cognate tRNAPhe. For mapping eRF1 regions cross-linked to mRNA

analogues, we used selective CNBr cleavage of the modified eRF1 after Met residues. The

major labeled product of cross-linking was isolated by SDS-PAGE and then treated with

hydroxylamine, endoprotease GluC or Arg-C with subsequent SDS-PAGE analysis of the

resulted labeled products. We found that all mRNA analogues used cross-linked with amino

acids in positions 31-33 in N-domain of the eRF1. These amino acids belong to motif GTx

conserved in eRF1 of all eukaryotes. The cross-linking results are in a good agreement with

data on modeling of eRF1 structure in 80S ribosomal termination complex by using a three-

dimensional structure similarity method.

This work was supported by Russian Foundation for Basic Research (grants 08-04-00508 to

GK) and by the grant from the Presidium of Russian Academy of Sciences (Program on

Molecular and Cell Biology) to G.K.

34

Poly(ADP-ribose) polymerase 1 is a key regulator of damage processing in

base excision repair

Khodyreva S.N., Sukhanova M.V., Ilina E.S., Kutuzov M.M., Lavrik O.I.

Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk, Russia

Poly(ADP-ribose) polymerase 1 (PARP1), functioning as DNA nick-sensor, interacts

with base excision repair (BER) DNA intermediates containing single-strand breaks. Bound

to DNA breaks, PARP1 catalyzes synthesis of poly(ADP-ribose) covalently attached to some

nuclear proteins and itself. Autopoly(ADP-ribosyl)ation of PARP1 facilitates its dissociation

from DNA breaks and is considered as a factor regulating DNA repair. PARP1 was identified

among the BER proteins cross-linked to the photoreactive branch-point BER DNA

intermediate along with apurinic/apyrimidinic endonuclease 1 (APE1), DNA polymerase β

(Pol β) and flap endonuclease 1 (FEN1). By functional assays in reconstituted systems, as

well as in cell extracts, we demonstrated that PARP1 and its poly(ADP-ribosyl)ation is

involved in regulation of activity of the base excision repair enzymes – APE1 (3’-5’

exonuclease activity), Pol β, FEN1. PARP1 and its poly(ADP-ribosyl)ation was shown to

more efficiently influence DNA synthesis in long patch BER. PARP1’s ability to interact with

intact AP sites and AP sites processed by APE1 via covalent via Schiff base intermediate was

demonstrated in cell extracts and with pure PARP1. The identity of PARP1 as the target for

cross-linking to AP sites in cell extracts was proved by MALDI-TOF-MS analysis. PARP1

does not cleave AP sites, but instead forms a stable intermediate with this lesion. Thus, in

addition to well-known role of PARP1 as nick-sensor, we demonstrated its interaction with

DNA intermediate of the BER process at the stage preceding incision of sugar-phosphate

backbone of DNA. Interaction of PARP1 with AP sites, along with the previously detected

interaction with DNA breaks, demonstrates PARP1’s role as a key sensor of lesions appeared

in the BER process. PARP1 is able to interact with AP sites cleaved by APE1 and in the

absence of Pol β, when the removal of 5' deoxyribose is failed, interaction of PARP1 with this

intermediate stimulates synthesis of poly(ADP-ribose) polymer, which is known as a death

signal.

Acknowledgements: This work was supported by RFBR, project № 10-04-01083,

Program of RAS “Molecular and Cellular Biology”

35

Oxothiomolybdenum derivative of the crown heteropolyanion {P8W48}:

Structures, studies in solution and electrochemical properties.

Korenev V.S.a,b, Floquet S.a, Marrot J.a, Mbomekallé I.-M.,a Haouas M.,a Taulelle F.,a

Cadot E.a

a Institut Lavoisier de Versailles, UMR 8180, Université de Versailles, 45 avenue des Etats Unis, 78035 Versailles, France.

b Nikolaev Institute of Inorganic Chemistry, Novosibirsk 90, Russia.

Polyoxometalates constitute a wide family rich of more than several thousand inorganic

compounds displaying various properties in catalysis, medicine, magnetism, conductivity,

analytical or supramolecular chemistry. Coordination of the oxothiocations [Mo2O2S2]2+ or

[Mo3S4]4+ with various vacant POMs constitutes an efficient approach to design inorganic

materials and some supramolecular systems, sometimes spectacular, had been obtained by

following this strategy [1-4]. This poster highlights the synthesis and the characterization in

the solid state and in solution of a new PolyOxoThioMetalate combining 4 oxothiocations

[Mo2O2S2]2+ with a cyclic P8W48 POM. The structure of the compound is described and the

solution studies carried out by UV-Vis. spectroscopy. Formation of two isomers is shown by 31P NMR studies.

[1] Cadot, Pilette, Marrot, Sécheresse, Angew. Chem. Int. Ed., 2003, 42, 2173

[2] Marrot, Pilette, Sécheresse, Cadot, Inorg. Chem., 2003, 42, 3609

[3] Cadot, Béreau, Marg, Halut, Sécheresse, Inorg. Chem., 1996, 35, 3099.

[4] Duval, Pilette, Marrot, Simonnet-Jégat, Sokolov, Cadot, Chem. Eur. J., 2008, 3457-3466.

36

The structure of the SBP2 binding site on the large subunit of the human ribosome

Kossinova O.A.1,2, Malygin A.A.1, Krol A.2, Karpova G.G.1

1Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian

Academy of Sciences, Novosibirsk, 630090, Russia; 2Architecture and Reactivity of RNA,

Strasbourg University, CNRS, IBMC, 15 rue René Descartes, 67084 Strasbourg, France

The biological form of the trace element selenium is amino acid selenocysteine. It is

encoded by an UGA triplet (Sec codon), which acts generally as a stop codon, and is

incorporated into selenoproteins during translation by a specialized machinery. The exact

mechanism of selenocysteine insertion has not been established yet, nevertheless, it is known

that the process involves a specific stem-loop located in the 3’-UTR of selenoprotein mRNAs,

termed as Selenocysteine Insertion Sequence (SECIS), and some protein factors. One of these

factors is SECIS RNA Binding Protein 2 (SBP2), which is necessary for ribosomal

recognition of the UGA triplet as the Sec codon.

Earlier, SBP2 was found to bind specifically to 60S ribosomal subunit. Here, using

direct UV-induced crosslinking or bifunctional crosslinking reagents (diepoxybutane or 2-

iminothiolane), we have shown that SBP2 contacts 28S rRNA. Besides, crosslinking with 2-

iminothiolane showed that SBP2 neighbors proteins L4 and L27a on the 60S subunit.

Ribosomal protein(s) L7/L7a/L8/L13/L19 are also potential neighbors of SBP2 as revealed

from diepoxybutane cropsslinking.

To investigate molecular contacs of SBP2 in the course of translation of mRNAs,

coding for selenoproteins, we used a model mRNA consisting of short 5’-UTR followed by

the sequence coding for tetrapeptide Met-Sec-Phe-Phe, spacer sequence and SECIS-element

at the 3’-end. It was shown that SBP2 is bound to the SECIS-element in the 48S pre-initiation

complex and in the 80S post-translocation complex in a cell-free protein synthesizing system

from rabbit reticulocytes.

This work was supported by Russian Foundation of Basic Research (grant 08-04-00508-a to G.K.) and by the Presidium of RAS (Program “Molecular and Cell Biology”).

37

Complexation and extraction of non-ferrous metals by calixarenes, upper

and lower rim functionalized with PO-groups.

Torgov V.1, Kostin G.1, Us T.1, Korda T.1, Kalchenko V.2, Arnaud-Neu F.3

1 Institute of Inorganic Chemistry SB RAS, Russia, Novosibirsk 2 Institute of organic chemistry Ukrainian NAS, Ukraine, Kyiv 3 Institut Pluridisciplinaire Hubert Curien, France, Strasbourg

In contrast to other macrocyclic compounds, calixarenes have two sites of modification

(upper and lower rim) with different distance between donor groups placed at these sites that

can be used for additional tuning of spatial fitting between ligand and metal cation.

Calix[n]arene-phosphineoxides with PO-groups in different sites (L) were compared in

the extraction of non-ferrous metals (M2+ = Zn2+, Co2+, Ni2+, Cu2+). The stoichiometry of

mono- and binuclear complexes [Mm(NO3)2mLn] (m, n = 1,2) was defined by extraction

methods and extraction constants were calculated. During the extraction calix[4]arenes

grafted at the lower rim with four phosphine oxide groups form most stable 1:1 complexes

with M2+ due to the proximity of donor groups and better geometrical fitting ligand-cation. At

the same time the upper rim grafted calix[4]arene tetraphosphine oxides are more inclined to

formation of 1:2 and 2:1 complexes. Extractions constants of [ML(NO3)2] increase from

upper to lower rim derivatives due to the shorter distance between donor groups and better

spatial fit to small M2+ cations. Higher steric flexibility of lower rim modified L after

dealkylation of upper rim results in decrease in extraction constants. Similar influence of

steric effects was found for flexible calix[6]arenes and after increase of spacer length in

calix[4]arenes.

In complexes M2L with upper rim modified calix[4]arenes each metal cation is

coordinated by two PO groups and two nitrate anions while for lower rim modified

calix[4]arene complexes unusual zwitter-ionic structure was determined. In that structure one

M2+ cation is coordinated with three PO-groups and one nitrate anion and another M2+ is

coordinated with one PO and three bidentate NO3- ligands.

The work was supported by ISTC-3405 and RAS project № 5.8.2.

38

New inclusion compounds of molecular container cucurbit[8]uril

Kovalenko E., Gerasko O., Fedin V.

Nikolaev Institute of Inorganic Chemistry, SB RAS, 630090, Russia, Novosibirsk, Lavrentiev

Ave. 3; e-mail: e.a.kovalenko@niic.nsc.ru

The growing interest to inclusion compounds is essentially caused by the unique

microenvironment provided by the host cavity to a guest metal complex which is

reminiscent of natural enzymes. Encapsulation into cavitands may be the best way to

isolate and stabilize unusual oxidation states and coordination environments of metals and

may completely change the guest’s properties. Cucurbit[8]uril (CB[8], C48H48N32O16) is

one of the most fascinating hosts, a rigid barrel-shaped molecule with two hydrophilic

carbonyl-fringed portals and hydrophobic inner cavity of

8.8 Å diameter.

We have developed synthetic procedures for

preparation of СВ[8] inclusion compounds with Au, Pt,

Pd, Ru complexes containing cyclic or acyclic aliphatic

polyamines like en, dien, and cyclam as ligands in high

yields. The products were characterized both in solid

state and in aqueous solution by X-ray single crystal analysis (for example, the structure of

{trans-[Ru(en)2Cl2]@CB[8]}+ is shown on figure), IR, UV, ESI-MS and TGA.

It has been shown that inclusion into the cavity of cucurbit[8]uril is helpful for

stabilization of guest complexes towards thermolysis, isomerizations etc.

This work was supported by RFBR (grant № 08-03-00088) and by RAS Gr. № 5.6.1,

SB RAS Gr. № 107.

39

Dramatic conformation differences in a brain-specific RNA between chimpanzee and humans. A link to human brain evolution?

Beniaminov A.1,2, Westhof E.1 and Krol A.1

1Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow (Russia) 2Institute of Molecular and Cellular Biology, Centre National de la Recherche Scientifique, Strasbourg (France). a.krol@ibmc-cnrs.unistra.fr

The human genome encodes about 20,000 genes, representing only 1.5% of its

sequence. To establish whether functional elements can exist in the “non-coding” remaining

part (i.e. regions lacking protein-coding capacities), Pollard et al. (Nature 443, 167-172, 2006)

undertook a genome-wide bioinformatic approach by aligning whole genome sequences from

a variety of vertebrates. They identified 49 regions (termed Human Accelerated Regions or

HARs) showing a significantly accelerated rate of nucleotide substitutions in humans since

the divergence from our common ancestor with chimpanzee. The 118 base pair HAR1 region

exhibits the highest rate of acceleration with 18 substitutions compared to chimpanzee, HAR1

being well conserved in other vertebrates since only two nucleotide changes occurred between

chicken and chimpanzee. To our surprise, and in contrast with the Pollard data, we found by

RNA structure mapping in solution and other experimental approaches that the human and

chimpanzee HAR1 RNAs adopt radically different 2D structures: a stable cloverleaf in the

case of humans whereas the chimpanzee counterpart folds into a less stable, extended hairpin.

However, computer predictions proposed for chimpanzee the same structure as in humans

(supported by base covariations) although we never detected it in our experiments. We

hypothesized that the cloverleaf structure is the functional conformation and that a ligand

(protein or RNA) helps the chimpanzee-hairpin to switch to the cloverleaf one. To verify the

hypothesis, brain protein extracts were searched by chromatography affinity/mass

spectrometry. Very interestingly, we could isolate protein hnRNP L as an interactant to the

chimpanzee but not to the human HAR1 RNA. Further experiments are underway to

substantiate this finding.

It was shown by others that HAR1F RNA is highly expressed in the developing human

neocortex (which is especially well developed in humans) and co-expressed with reelin, a

protein that is crucial in specifying the six-layer structure of the human cortex. Also, down

expression of HAR1 RNA was observed in patients with Huntington disease. Altogether,

these findings strongly suggest that the rapid changes that occurred in HAR1 are linked to

human brain evolution.

40

Lipophilic siRNA conjugates: cellular uptake and anti-MDR activity

Kruglova N. S., Meschaninova M. I., Venyaminova A. G., Zenkova M. A., Vlassov V. V.,

Chernolovskaya E. L.

Institute of Chemical Biology and Fundamental Medicine SB RAS, 8 Lavrentiev ave., Novosibirsk 630090, Russia

siRNAs are considered to be promising therapeutic agents for sequence-specific

silencing of disease-related genes. However, the problem of inefficient delivery of siRNAs

into cells limits their biomedical application. Conjugation of siRNA to the molecules, which

can be internalized into the cell by natural transport mechanisms, can result in the

enhancement of siRNA cellular uptake.

In this work we investigated the carrier-free accumulation of nuclease-resistant siRNA

equipped with lipophilic residues tethered on the 5'-end of the sense strand in HEK293,

HepG2 and KB-8-5 cancer cells. We showed that conjugates of siRNA with cholesterol and

oleyl-lithocholic acids effectively penetrated (up to 100 %) into the cells at siRNA

concentrations: 0.2 – 10 mkM. Whereas the uptake of oleic or lithocholic acid siRNA

conjugates was insignificant and comparable to that of the unmodified siRNA. We found that

efficacy of cellular uptake enhanced when the length of amino-hydrocarbon linker between

siRNA and lipophilic residue increased. The conjugates of siRNA and cholesterol tethered

with aminohexyl or aminododecyl linkers demonstrated the best transfection properties

regardless of cell type. The mean values of fluorescence of samples treated by conjugate of

siRNA and cholesterol bound aminohexyl linker were approximately in 1.4 – 4 times higher

in comparison with that of the similar conjugate with aminopropyl linker. The biological

activity of cholesterol-conjugated siRNAs targeted to MDR1 gene was tested in KB-8-5 drug

resistance cell line. Incubation of the cells in the presence of the conjugates and 300 nM

vinblastin resulted in cell death. In 6 days after treatment of the cells with the conjugate of

siRNA and cholesterol tethered with aminohexyl linker only 45 % of living cells was

observed. Thus, we developed the optimal structure of anti-MDR1 siRNA-lipohilic

conjugates. These conjugates are able to penetrate into cells of different types without

transfection reagents, to silence the expression of the target gene and to reverse the multiple

drug resistance of cancer cells making their susceptible to chemotherapy.

This work was supported by RAS programs “Molecular and Cellular Biology” and “Basic sciences for medicine”, RFBR 08-04-01073, grant from SB RAS No. 41, FCP No. 02.512.11.2294.

41

Study of supramolecular machines of nucleotide excision repair by

using chemical approaches Lavrik O.I., Krasikova Y.S., Rechkunova N.I.

Institute of Chemical Biology and Fundamental Medicine, Novosibirsk, Russia

The nucleotide excision repair (NER) is one of the major repair systems to remove a

wide range of helix distorting lesions from DNA, including those formed by UV light, various

environmental mutagens and certain chemotherapeutic agents. Defects in NER are associated

with several human autosomal hereditary diseases. The coordination of the assembly of the

NER complexes and the sequential individual reactions is achieved through multiple DNA-

protein and protein-protein interactions. We have studied two key stages of global genome

repair (GGR), namely, damage recognition and assembly of preincision complex. The new

technique to study assembly of NER machine was elaborated. The interaction of key protein

factors of the NER process, XPC-HR23B, XPA, and RPA, with DNA structures that mimic

NER intermediates has been analyzed. Using DNA duplexes containing photoreactive 5I-

dUMP residues in the certain positions either in damaged or in undamaged strands and

fluorescein group linked to uridine residue as the lesion, direct evidence of preferential

contacts of XPC-HR23B, the damage sensing factor of GGR, with undamaged strand was

provided. The photocrosslinking positioning of XPC-HR23B on damaged DNA is in

accordance with the X-ray data for Rad4. RPA was shown to contact mainly with the 5' side

of undamaged strand; however efficient crosslink with damaged strand was also detected.

XPA shows two maximums of crosslinking intensities located on the 5’ side from a lesion.

Very similar results were obtained for damaged DNA with 15 nt bubble. These results show

for the first time the localization of XPA and RPA in the 5’ side of the lesion and together

with other results suggest their key roles for positioning the NER preincision complex. The

findings support the mechanism of loading of the structure specific endonuclease ERCC1-

XPF by XPA on the 5’ side from the lesion before damaged strand incision. This scenario is

in agreement with reported 5’ DNA strand incision by ERCC1-XPF prior to the 3’ DNA

strand incision by XPG. Together XPA and RPA would play a structural role and ensure a

proper three-dimensional structure of the DNA intermediate for excision in addition of being

involved in the DNA damage strand recognition. Therefore affinity labeling technique is

advantageous approach to study architecture of DNA repair complexes.

This work was supported by the Russian Foundation for Basic Research, grant no. 10-

04-00837 and by grant from RAS program on Molecular and Cellular Biology.

42

Sensor materials based on cyclodextrins immobilized on the silica

microspheres. Fluorescence and spin-label ESR study. Livshits V.A., Voronina L.V., Demisheva I.V., Alfimov M.V.

Center of Photochemistry, Russian academy of sciences, Moscow, Russia

New sensor materials based on the charged cyclodextrin polymers adsorbed on the

modified silica microspheres, and monomer cyclodextrin derivatives covalently attached to

the silica microspheres were prepared. The binding isotherms and dependences of the CD-

polymer binding on the incubation time in solution and ionic strength were studied. The

volatile chemicals, toluene and naphthalene, were detected by their adsorption on the above

materials using the fluorescence method. Both bound hydrocarbons display monomer and

excimer fluorescence, their relative intensities and maximum positions are dependent on the

type of CD receptor. The adsorption of the negatively charged fluorophores, dansylglycine

(DG) and 2,6-ANS, in aqueous solution was also studied. Fluorescence spectra of the

adsorbed fluorophores are shifted up to 150 nm to the short wavelengths as compared with

aqueous solutions and CD complexes in solution.

It was found that the excitation of the naphthalene adsorbed on the microspheres with the

CD-complexed DG results in the fluorescence energy transfer from naphthalene to DG. This

phenomenon may be used for studying the spatial distribution of the adsorbed fluorescent

molecules and also a selective detection of naphthalene.

In order to investigate the molecular dynamics and character of binding of analytes in the

CD-based chemosensors the spin probes of different structure, form and electrical charge

were used as analyte analogues. The conventional β-CD and β-CD modified with phenyl

residues were used as host macrocycles which were attached via spacer group to silica

microspheres. It is shown that most CD-complexes in both types of microparticles exist in

two conformational states which have having quite different rotational mobility and polarity

of local environment: These states are assigned to (1) the complexes which additionally non-

covalently adsorbed on the silica surface and (2) the desorbed complexes which rotate around

several ordinary bonds relative to the silica particle. The rotational diffusion coefficients and

their activation energies were estimated for both states of complexes and for different spin

probes.

This work was financially supported by RFFI, grant 10-03-01166a.

43

Key role of human ribosomal protein p40 in the recognition of the

hepatitis C virus Internal Ribosome Entry Site

Malygin A.A.1, Kossinova O.A.1, Shatsky I.N.2, Loktev V.B.3 and Karpova G.G.1

1Institute for Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian

Academy of Sciences, Novosibirsk, 630090, Russia, 2Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow,

119992, Russia, 3State Research Center of Virology and Biotechnology “Vector”, Koltsovo, Novosibirsk region,

630559, Russia

Ribosomal protein (rp) p40, also known as a 37 kDa laminin receptor precursor, is a

structural element of the small subunit of the human ribosome. This protein is located on the

back side of the subunit contacting with its head and body. The N-terminal and central parts of

p40 are homologous to prokaryotic rpS2, whereas its C-terminal domain (CTD) responsible for

laminin binding is specific for eukaryotes. Content of p40 in ribosomes is not constant, it

depends on cellular status and increases upon active cell growth and polysomes formation.

We have shown that preparations of 40S ribosomal subunits isolated from full-term human

placenta specimens are deficient in p40 to a varying extent. We found that recombinant p40 can

saturate these 40S subunits up to the equimolar content. Using a set of p40 truncated mutants

with the serial deletions in the CTD, we have shown that this domain plays a crucial role for the

protein binding to 40S subunits. We have found that the 40S subunits saturated with p40 have

higher affinity to the Internal Ribosome Entry Site (IRES-element) of the hepatitis C virus

(HCV) RNA as compared to the p40-deficient subunits. Monoclonal antibodies 4F6 specific to

p40 CTD prevented HCV IRES binding to 40S subunits and blocked translation of IRES-

containing RNA in a cell-free translation system. The results indicate that p40 is involved in the

binding of the HCV IRES to the ribosome and, therefore, in translation initiation of HCV RNA.

A possibility of involvement of the CTD of p40 in HCV IRES binding to the 40S subunit is

discussed.

This work was supported by grant from the Russian Fund for Basic Research (grant No. 08-

04-00593-а to A.M.) and Presidium of the Russian Academy of Sciences (Program on Molecular

and Cell Biology).

44

Gas hydrates and ionic clathrate hydrates: recent results

Manakov A.Yu.1,2, Komarov V.Yu.1,2, Rodionova T.V.1, Terekhova I.S.1, Ogienko A.G.1,

Grachev E.V.1, Ildyakov A.V.1, Burdin A.A.1, Stoporev A.S.2, Kutaev N.V.2

1 Nikolaev Institute of Inorganic Chemistry SB RAS, Akad. Lavrentiev ave., 3, Novosibirsk, 630090, Russian Federation

2 Novosibirsk State University, Pirogova Str. 2, Novosibirsk, 630090, Russian Federation

Clathrate hydrates are crystalline inclusion compounds in which the host framework is

formed by water molecules connected through hydrogen bonds. The cavities of the

framework are occupied by the guest molecules of appropriate size and molecular shape; the

host-guest interactions in true clathrate hydrates are purely van-der-Waals. In the crystal

structure of ionic clathrate hydrates water molecules and (most often) guest anions build up a

polyhedral hydrogen-bound framework; its cavities are occupied by cations. The most typical

representatives of ionic clathrate hydrates are polyhydrates of the salts of alkylonium cations

with monoatomic halide anions. Short review of the recent results of our group (Clathrate

compounds laboratory, NIIC SB RAS, Novosibirsk) will be given in this presentation.

In the first part of the presentation we will discuss some new data concerning

structures and stoichiometrys of high-pressure gas hydrates Distinctive feature of high-

pressure gas hydrates is formation of new types of gas hydrate frameworks including

frameworks with space-filling polyhedrons and ice-like non-polyhedral frameworks. New

types of frameworks of this type were generated with use of original algorithm developed by

our group. In addition, some new data concerning multiple occupation of gas hydrate cavities

by guest molecules will be considered. Second part of the presentation will be dedicated to

discussion of some new features of crystal structures of ionic clathrate hydrates of

tetrabutilammonium halogenides.

45

Control of ribozyme folding avoids cell death

Beckert B.1,2, Hedegaard M.M.2, Nielsen H.2 and Masquida B.1

1 IBMC-CNRS, Architecture et Réactivité de l’ARN, Université de Strasbourg, Strasbourg,

France 2 Department of Cellular and Molecular Medicine, The Panum Institute, University of

Copenhagen, Copenhagen, Denmark

Most ribozymes fold directly into their active conformation and execute their function promptly. Group I introns, for example, generally fold into their catalytically active conformation during transcription and splice out at an early stage of processing of the precursor to leave the flanking exons ligated. Structural similarities with group I ribozymes may suggest that the GIR1 branching ribozyme also folds directly into an active conformation. GIR1 biological function is to provide the downstream mRNA encoding a homing endonuclease (HE) with a lariat cap, a substitute for a conventional mRNA cap. However, since GIR1 together with the HE mRNA are embedded in a ribosomal RNA group I intron (GIR2) and GIR1 catalyzes a branching reaction (resulting in a cleavage of the pre-mRNA), early cleavage would preclude correct rRNA processing.

Thus, the GIR1 branching ribozyme potentially represents a serious threat to the host organism because the outcome of processing of the ribosomal precursor depends on an intricate balance between the rate of transcription, folding and activity of the splicing and branching ribozymes of this twin-ribozyme intron. If GIR2 acts prior to GIR1, splicing occurs resulting in ribosomal RNA exons ligation. On the opposite if GIR1 cleaves first, GIR2 is rendered inactive and the ribosomal RNA is lost. This pathway has been shown to predominate during starvation-induced encystment.

The aim of the present study is to reveal the mechanism that keeps GIR1 activity in check during normal growth such that ribosomal RNA is formed. By a combination of native gel electrophoresis and Fe-EDTA probing we identified a domain that undergoes conformational switching resulting in an inactive or an active state of the ribozyme. Modelling based on structure probing suggests that the active form is characterized by docking of a domain (P2P2.1) that serves to stabilize the core while the inactive form sequesters parts of this domain in a separate hairpin structure (HEG P1). Intriguingly, the very same switch was recently found to operate in reverse in the release of the HE mRNA following lariat cap formation. It appears that one aspect of adaptation of the group I scaffold to its new role in branching is the replacement of the universal L9/P5 tertiary interaction, that tethers the primary domains of group I introns, by a conformational switch that allows host functions to take control of the GIR1 ribozyme.

46

Effect of Cucurbit[7]uril on the Primary Photoprocesses of

Thiacarbocyanine Dye Atabekyan L.S., Petrov N.Kh., Ivanov D.A., Chibisov A.K.

Photochemistry Center of the Russian Academy of Sciences, Moscow

Cyanine dyes represent an important class of organic synthetic dyes which exhibit unique properties both in the ground and the excited states. In the ground state the dyes reveal an ability to form dimers, H- and J-aggregates whereas in the excited singlet state cyanines appear to exhibit fluorescence and trans – cis isomerization. The quantum yield of the intersystem crossing is usually very low. Since electron transfer mostly occurs in the triplet state of cyanines in solution the finding of conditions of the triplet state population is of keen interest. The binding of cyanines with cucurbit[n]utils due to host-guest complexation might be promising to restrict the efficiency of radiationless deactivation of cyanines and to control the pathways of the singlet and triplet decay. Here we present the results on ns-laser photolysis and fluorescence study of 3,3-diethylthiacarbocyanine dye (Cy) in the presence of cucurbit[7]uril (CB7) in aqueous solution. Peak at 515 nm (Fig.1) which becomes pronounced in the presence of CB7 may be attributed to Cy dimers. Binding of Cy with CB7 also results in the five fold enhancement of fluorescence of the dye. Upon laser excitation (532 nm) of air -free aqueous solution of Cy in the presence of CB7 a few short lived transients were found (Fig. 2).

400 500 6000.0

0.5

1.0

1.5

2.0

nm

А1

2

400 500 600 700-0.2

-0.1

0.0

0.1

nm

3e-5 9e-5 30e-5

A

Figure 1. Absorption spectra of Cy in the presence (2) and absence (1) of CB7.

Figure 2. Time-resolved absorption spectra upon ns-laser photolysis of Cy in the presence of CB7.

Transient absorption originates from the monomer dye triplet > 630 nm, the dimer dye triplet peaking at 540 nm in the difference spectra, as well as radicals as the product of one-electron oxidation (460 nm) and one-electron reduction (420-430 nm) of the dimer.

The work was supported from the program № 6 of basic research of chemical department of RAS.

47

[Co(sep)]3+ [Co(dipy)3]3+ [Ru(dipy)3]2+

[Zn(dipy)3]2+ pH=8

[Zn(dipy)3]2+ pH=11

TCAS

Conformational transitions of p-sulfonatothiacalix[4]arene depending on

guest size and pH value of solutions Skripacheva V.V.a, Mustafina A.R.a, Gruner M.b, Gubaidullin A.T.a, Katsyuba S.A.a,

Zvereva E.E.a, Kleshnina S.R.a , Soloveva S.E.a, Habicher W.b,. Konovalov A.Ia

aA.E. Arbuzov Institute of Organic and Physical Chemistry, Arbuzov street, 8, 420088,

Kazan, Russia, bInstitute of Organic Chemistry, Technical University, Bergshtrasse street, 66, 01069,

Dresden, Germany

The supramolecular chemistry of water-soluble calixarenes is widely investigated

because of the diversity of such molecules in formation of inclusion and coordination

complexes in both solution and the solid state. p-Sulfonatothiacalix[4]arene (TCAS) is a

unique molecular bilding block to construct versatile supramolecular assemblies in the

presence of suitable guest molecules.

Kinetically inert cobalt

(III) and ruthenium (II)

complexes interact with

TCAS cavity giving

inclusion complexes, with

the complexation mode

being independent on pH,

while the variation of the conformation of TCAS with the guest size has been revealed.

According to X-ray data cobalt sepulcrate (small complex) is bound with cone shaped TCAS.

On going to more bulky tris-dipyridyls of cobalt and ruthenium TCAS adopts pinched cone

conformation. It is shown by 1D and 2D NMR techniques in solution and X-ray data in solid

state that the interaction with larger sized and kinetically labile zinc tris-dipyridyl provide the

versatile pH-dependent effect on the TCAS conformation. So, TCAS in cone conformation

binds zinc trisdipyridyl complex in neutral media in the outer-sphere mode, the formed outer-

sphere associate has a low stability. The deprotonation of phenolic groups of TCAS at pH 8

favours the partial cone conformation of TCAS, resulting in the inclusion of zinc complex.

The pH increase up to 11 results in complex formation between TCAS in 1,2-alternate

conformation and dechelated zinc complex with inner-sphere coordination of zinc ion by

phenolate groups and sulfur atom in stoichiometric ratio 1:2 correspondingly.

48

Searching translational operator for streptomycin mRNA of E. coli.

Surdina A.V.1, Golovin A.V.2, Rassokhin T.I.3, Spiridonova V.A.3, Kopylov A.M.1,3

1 Chemistry Department of Lomonosov Moscow State University,

2 Department of Bioengineering and Bioinformatics of Lomonosov Moscow State University, 3 A .N. Belozersky Institute of Physical Chemical Biology of Lomonosov Moscow State

University

In E. coli cells ribosomal small subunit biogenesis is regulated by RNA-protein

interactions involving proteins S4 and S7. S7 initiates assembly of the subunit head

interacting with the 3’ major domain of the 16S rRNA. During shift-down of rRNA

synthesis, an excess of S7 inhibits its own translation by interacting with intercistronic

region of streptomycin (str) mRNA between cistrons S12 and S7, which is 96

nucleotides long.

Many bacteria do not have the extended intercistronic region challenging

development of specific approaches for searching putative mRNA translational operon,

abling to interact with proteins.

We applied SERF approach (Selection of Random RNA Fragments) to reveal

regulatory regions of str mRNA. By random hydrolysis a set of random DNA fragments

has been generated from str operon, and then transcribed into RNA; the fragments being

able to bind protein S7 (serfamers) have been selected by iterative rounds. S7 binds to

single serfamer, 109 nucleotide long (RNA109), derived from the intercistronic region.

After multiple copying and selection, the intercistronic mutant (RNA109) has been

isolated; it has enhanced affinity to S7. RNA109 binds to the protein better than

authentic intercistronic str mRNA; apparent dissociation constants are 26 +/- 5 and 60

+/- 8 nM, respectively.

Location of S7 binding site on the mRNA, as well as putative mode of regulation

of coupled translation of S12 and S7 cistrons have been hypothesized.

The work has been supported by RFBR 08-04-01540-а, 09-04-90467

49

ISIDA approaches to virtual screening based on fragment and

pharmacophoric descriptors Varnek A.

Laboratory of Chemoinformatics, UMR 7177 CNRS, University of Strasbourg

http://infochim.u-strsabg.fr

Virtual screening is a process in which large libraries of compounds are automatically

evaluated using computational techniques. Its goal is to discover putative hits in large

databases of chemical compounds (usually ligands for biological targets) and to remove

molecules predicted to be toxic or those possessing unfavorable pharmacodynamic or

pharmacokinetic (ADME) properties. In drug design, two types of virtual screening are

known: structure-based and ligand-based. The former explicitly uses the three dimensional

structure of a biological target, whereas the latter uses only information about structure of

organic molecules and their properties (activities). This presentation concerns recent

developments in two main approaches to ligand-based virtual screening - similarity search

and SAR/QSAR –modeling - integrated into the ISIDA (In SIlico design and Data Analysis)

program. New types of molecular descriptors, original machine-learning techniques and

algorithms and their practical application to model different biological activities will be

discussed. Particular attention will be paid to the WEB-based platform for virtual screening

integrating developed SAR/QSAR models and tools and freely available for the users at

http://infochim.u-strasbg.fr/webserv/VSEngine.html.

References: A. Varnek, D. Fourches, D. Horvath, O. Klimchuk, C. Gaudin, P. Vayer, V.

Solov’ev, F. Hoonakker, I. V. Tetko, G. Marcou “ISIDA - Platform for virtual screening

based on fragment and pharmacophoric descriptors”

Current Computer-Aided Drug Design, 2008, 4 (3), 191-198.

50

Supramolecular complexes of nucleic acids: new materials, devices and

biotherapeutics Vlassov V.V.

Institute of Chemical Biology and Fundamental Medicine SB RAS

Oligonucleotides can serve as multifunctional building blocks for design of different

materials, nanomachines and therapeutics targeted to specific biopolymers and specific cells.

Antisense oligonucleotides, aptamers and double-stranded small interfering RNAs have

become important instruments for manipulating gene expression in biological experiments.

These nucleic acids are considered as a promising gene targeted therapeutics.

Approaches of supramolecular chemistry are used to develop the therapeutic

compositions capable of efficiently delivering nucleic acids in eukaryotic cells. One approach

is based on design of supramolecular complexes containing therapeutic nucleic acids

associated with polycations or cationic lipids. We have designed new cationic lipids and

cholesterol conjugated polyethyleneimines that display low toxicity and provide efficient

compactization and delivery of both large DNAs and small oligonucleotides and siRNAs in

different mammalian cells.

We have found that affinity of oligonucleotides to cellular membrane can be enhanced

by assembling oligonucleotide molecules into supramolecular complexes that form multiple

weak bonds with the lipid surface of the membrane. An approach to prepare supramolecular

complexes of variable sizes and methods to equip these complexes with cholesterol residues

providing additional affinity to the membranes have been developed. Complexes if siRNA,

bearing cholesterol on the sense strand, are efficiently taken up by different cancer cells, do

not cause any toxic effects and the incorporated nucleic acids efficiently inhibit expression of

the target genes.

This research was supported by the Russian Academy of Science under the programs “Molecular and Cell Biology”, “Science to Medicine”, “Nanotechnology and Nanomaterials”, and the program for support of leading scientific schools (grant no. NSh-7101.2010.4), Russian Foundation for Basic Research (grant no. 08-04-00753a), and Ministry of Science and Education of the Russian Federation (state contract no. 02.512.11.2200 02.512.11.2294 ). 1. Gusachenko (Simonova) O.N., et al. // Human Gene Therapy. – 2008. - V. 19. - № 5. - P. 532-546 2. Gusachenko (Simonova) O., et al. // Journal of Biomaterials Science. - 2009. - V.20. - P.1091-1110. 3. Medvedeva D.A // J. Medicinal Chemistry. - 2009. - V52 - N.11.- Р. 6558-6568.

51

Supramolecular chemistry of nitronyl and imino nitroxyl radicals and cucurbit[n]urils

Vostrikova K., Fedin V.

Nikolaev Institute of Inorganic Chemistry SB RAS, Novosibirsk

Since the characterization of its first member, cucurbit[6]uril in 1981, the cucurbit[n]uril (CB) (n = 5-10) family has rapidly expanded with the preparation of the higher homologues. Amongst systems exhibiting host properties, CBs are very attracting since they have a hydrophobic cavity accessible by two identical portals composed of a rim of polar ureido carbonyl groups. A large variety of molecular objects have been hosted in CBs. While the hydrophobic cavity is capable to accept different small lipophilic molecules, the polar portals coordinate various cations which are associated with high bonding constants. Owing to their rigid structure, CBs have found important applications as synthones for designing different assemblies: rotaxanes, molecular necklaces and dendrimers are examples of such

applications. A majority of CBs inclusion complexes

contains diamagnetic molecules as the guests.

Recently were published a series of works, where

the recognition properties of CBs with respect to

organic radicals were reported. Most paramagnetic

guests involved in these studies are nitroxyl radicals, mainly TEMPO derivatives, for which a

well documented synthetic chemistry allows to design guests suitable to a host under study.

Among the nitroxides, nitronylnitroxides (NNs) also benefit from easy structural variations and,

in contrast to TEMPO, they have a delocalized electronic structure. This property favors

intermolecular coupling pathways resulting in magnetic properties unprecedented for purely

organic species such as ferromagnetic ordering at low temperature. It is expected that using

NNs, the role of paramagnetic guests would not be restricted to that of a probing inclusion

processes. They might also play the role of synthones for designing high-spin molecules based

on host-guest chemistry and, more generally, molecular magnetic materials. Inclusion may favor

the aggregation of spin carriers or, in an opposite way, may result in an efficient screen for

cancelling out intermolecular magnetic interactions. In our report we present the investigation

results of inclusion of some nitronyl and iminonitroxyl radicals in cucurbit[n]urils, n = 7,8. In

this study a spectrophotometric titration was used for association constants estimation. The

solids were characterized by X-ray and thermal analysis. A theoretical support was used to

explain their interesting magnetic properties.

N

N

O

O

R

N

N

O

R

52

Non-enzymatic Recombination of RNA Zenkova M., Nechaev S., Lutay A., Vlassov V.

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Sciences, 8 Lavrentiev Avenue, Novosibirsk, Russia. 630090.

Studies of the last decades provided evidences that “there was once an RNA world” –

time period, when RNA polymers served as “carriers” of the genetic information and also provided some catalytic activities necessary for its replication. Regarding to the hypothesis, the primitive RNA molecules have to be synthesized in condensation reactions of the RNA monomers, then at the posterior stages, the RNA molecules had to be replicated via template-directed ligation reactions, in which Watson-Crick base pairing promotes the synthesis of the complementary sequence. One important obstacle of this pathway is an emergence of the novel sequences including catalytic ribonucleic acids, as far as not all sequences could be replicated via template-directed polymerization.

We assumed that novel RNA molecules could be formed in the recombination process consisting of two consecutive cleavage and ligation reactions. Both reactions can proceed in RNA molecules due to the presence of 2’-hydroxyl group in ribose and its ability to form 2’, 3’-cyclic phosphate. RNA fragments with 5’-hydroxyl group and 2’, 3’-cyclic phosphate, formed as a result of RNA cleavage, could cross-interact with each other forming new phosphodiester linkages and yielding new RNA molecules.

Here we describe results of our studies on the consecutive cleavage/ligation reaction of

short RNA-oligonucleotides and of the hundred nucleotides long fragments of two viral

RNAs, proceeding in the presence of magnesium ions. Cleavage/ligation of the

oligonucleotides was shown to occurre even in the absence of metal ions and its efficiency

demonstrated an escalating pH – yield profile within pH interval from 6.0 to 8.8, which

suggests base catalysis of the reaction proceeding via increasing of nucleophilic properties of

the attacking 5’-hydroxyl group. Divalent cations (Mg2+, Mn2+, Zn2+, Pb2+) accelerated

the reaction in pH-dependent manner. We found that at least 95% of the isolated products of

nonenzymatic ligation of short oligonucleotides were resistant to ribonuclease T1 under the

conditions, where 3’,5’-bonds are completely cleaved by the enzyme. The result of

comparative T1-probing can be attributed to the 2’,5’-bond formation in the nonenzymatic

ligation products. We show, that these consecutive cleavage/ligation reaction of the

fragments of viral RNAs results in the formation of the wide diversity of new products of

RNA recombination. We found that efficient RNA ligation occurs mostly in internal loops,

internal bulges and in stem structures, turned into stem-loops upon ligation.

Poster persentations

55

Photophysical Properties of Azacrown Styryl Dye and Its Complexes with

Metal Ions Atabekyan L.S., Lobova N.A., Vedernikov A.I., Gromov S.P., Chibisov A.K.

Center of Photochemistry of the Russian Academy of Sciences, Moscow

Photophysical properties of a new azacrown-containing 4-styrylpyridine (Dye) were studied

in the absence and presence of sodium, calcium, barium, silver(I) and lead(II) perchlorates in

acetonitrile at room and low temperatures. A big Stokes

shift was found for Dye in the absence (130 nm) and

presence of sodium, calcium and silver perchlorates (130-

150 nm) whereas for barium and lead perchlorates the

shift appeared to be 230 nm. This unusual behavior of fluorescence might be due to

formation of TICT state of the Dye molecule. Complexation with barium and lead

perchlorates results in strong blue shift (100 nm) of absorption band which is accompanied

by remarkable fluorescence enhancement (about 30 times). Along with the prompt

fluorescence both a delayed emission (P-type) at room temperature and phosphorescence at

77 K were found in the absence and presence of metal perchlorates. The lifetime of

phosphorescence is 10 ms and independent of metal ion, whereas the lifetime of delayed

fluorescence is 5 ms. Upon ns-laser photolysis of air-free solutions of the Dye and its

complexes with metal ions a short lived transient absorption was found. Quenching effect of

oxygen as well as the results on energy transfer with benzophenone as the triplet energy

donor imply in favor of the triplet nature of transient (Fig.1).

400 500 600 700 800-0.04

-0.02

0.00

0.02

0.04

nm

7e-6 30e-6 70e-6

A

0.0000 0.0004 0.0008

-0.02

-0.01

0.00

t, s

A

а)

0.0000 0.0004 0.0008

0.00

0.01

0.02

0.03

t, s

A

b)

Fig. 1. Time-resolved triplet-triplet absorption spectra of Dye in acetonitrile upon 352 nm laser pulse; a) time-course of the ground state recovery at = 510 nm, (b) triplet decay kinetics at = 640 nm.

The work was supported from RFBR (project № 09-03-00170) and the Russian

Academy of Sciences

O

N

O O

O

ONEt

ClO4

_

+

56

Conformational dynamics of antitumor drug onconase during the

interaction with RNA substrates

Alekseeva I.V.1, Koval V.V.1, Gabibov A.G.2, Fedorova O.S.1

1 Institute of Chemical Biology and Fundamental Medicine and Novosibirsk State University, Novosibirsk, Russia

2 Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia

Onconase (ONC), isolated from extracts of Rana pipiens oocytes, is a member of the

ribonuclease A superfamily that is toxic to cancer cells in vitro and in vivo. ONC is the

smallest, less catalytically efficient and more cytotoxic than most RNase A homologues. It is

very stable protein (Td = 890C, pH 6.0) and shows high resistance to the action of proteases at

physiological salt concentrations. The active site of ONC contains the catalytic triad (His10,

Lys31, and His97) that is characteristics of the RNase A superfamily.

We have applied stopped-flow kinetics to analyze the conformational dynamics of

specific (r(CUGGAG), dArUdGdA) and nonspecific (dArUdAdA) substrates during

processing of recombinant onconase at pH 7.4 and 6.0. These substrates were labeled with

FRET dye pair fluorescein/rhodamine. In substrate fluorescein was held in proximity to

rhodamine residue and its fluorescence was quenched. After the cleavage the fluorescence of

the substrate was increased 180-fold.

Monitoring fluorescence in stopped-flow experiments reveals at least three

conformational transitions in enzyme/substrate complex during the interaction of ONC with

ribooligonucleotide duplex within 50 ms - 100 s time ranges. These transitions reflect the

stages of enzyme binding to RNA and mutual adjustment of RNA and enzyme structures to

achieve catalytically competent conformation.

Only a single conformational change (at 70 s) is connected with nucleic acid cleavage.

This result provides evidence that several fast sequential conformational changes occur in

ONC after binding to its substrate, converting the protein into a catalytically active

conformation.

This work was supported by grants from the Russian Foundation of Basic Research

(10-04-00070), Russian Ministry of Education and Science (NSch- 3185.2010.4) and the

Siberian Branch of the Russian Academy of Sciences (21.22).

57

L36A-like protein is located in vicinity of the peptidyl transferase center of

the mammalian ribosome

Bulygin K.N.1, Baouz S.2, Woisard A.2, Le Caer J.-P.2, Hountondji C.2, Karpova G.G.1

1Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, pr.Lavrentjeva 8, 630090, Novosibirsk, Russia

2Institut Jacques Monod, Laboratoire de Photobiologie Moléculaire (CNRS-UMR 7033, BioMoCeTi) Universités Paris 6 et Paris 13, France

Protein environment of acceptor terminus of tRNA in the region of the peptidyl

transferase center (PTC) of the human ribosome was studied using 5’-[32P]-labeled analogues

of tRNAAsp bearing a zero length cross-linker at the 3’-terminus, namely, at either 3’-terminal

adenosine A76 of tRNAAsp (tRNAAsp(0)), or 3’-terminal cytidine C75 or C74 of the truncated

tRNA analogues (tRNAAsp(-1) and tRNAAsp(-2), respectively). tRNAAsp (0) was obtained

from yeast tRNAAsp by periodate oxidation of the 3’-terminal ribose to 2’,3’-dialdehyde. To

produce tRNA analogue truncated by one nucleotide, tRNAAsp was oxidated, treated with

lysine, dephosphorylated, purified by polyacrylamide gel electrophoresis and then oxidized

again. tRNA analogue truncated by two 3’-terminal nucleotides was prepared by repeating

this procedure twice. Stable tRNA-protein cross-links were obtained by treatment of

ribosomal complexes containing tRNA analogue and an mRNA with sodium

cyanoborohydride and resolved by denaturing gel electrophoresis in 8 М urea; their

formation completely depended on the presence of modified 2’,3’-dialdehyde nucleotide in

tRNA analogues. Cross-linked ribosomal proteins were identified by mass spectrometry

NanoLC-MS/MS. In all cases the only protein L36A-like was found to be cross-linked. The

yield of its modification was about 1% with tRNAAsp(0) and about 12% with tRNAAsp(-1) or

tRNAAsp(-2). Protein L36A belongs to the L44 family of ribosomal proteins. It has no

homologues in eubacteria but in archea its homologue is L44. It should be noted that in

modern three-dimensional structures of archaeon 50S subunits there are no proteins found

closer than 18 Å from PTC. Thus, we showed for the first time the presence of ribosomal

protein near the mammalian peptidyl transferase center.

58

Relation between side substituents and photo-chemical properties of

phthalocyanines

Chernonosov A.A.1,2*, Röder B.3, Solov'eva L.I.4, Luk'yanets E.A.4, Fedorova O.S.1

1Institute of Chemical Biology and Fundamental Medicine, Siberian Division of the Russian

Academy of Sciences, Lavrentyev Prospect 8, Novosibirsk 630090, Russia 2Institute of Human Ecology, Sovetskii Prospect 18, Kemerovo 650099, Russia

3Humboldt-Universität zu Berlin, Institut für Physik, Newtonstraße 15, 12489, Berlin, Germany

4Organic Intermediates and Dyes Institute, B. Sadovaya 1/4, Moscow 103787, Russia sandy@niboch.nsc.ru

*Corresponding author

Phthalocyanines (Pcs) have a great potential as photosensitizers in photodynamic

therapy (PDT) for the treatment of malignancies and other diseases. The chemical structure of

phthalocyanines can be modified by the introduction of substituents in the peripheral positions

of the tetraazaisoindole macrocycle, as well as by the coordination of metal ions with the

central nitrogen atoms. Primary goal of such modifications of Pcs is to tune the water

solubility and aggregation without significant changes in the photophysical properties.

In our work we report the spectroscopic and photo-physical characteristics of several

new Pc’s derivatives that can potentially serve as reagents for PDT. We examined Al(III) and

Zn(II) Pc-complexes having four carboxyl groups, the derivatives of these Pcs carrying one

long chain (oligonucleotide or minor groove binding ligand) and the Pcs having eight short

charged substituents in order to evaluate the spectroscopic and photo-physical effects of these

side residues on the chromophore properties.

The quantum yield of singlet oxygen generation, the fluorescence lifetimes, the

measurements of the triplet-triplet absorption, the decay-associated fluorescence spectra and

the transient absorption spectra, which are crucial in determining the feasibility of using any

dye as a reagent for PDT, are reported and linked to the structure of the substituents.

Supported by a Grant from the Russian Foundation for Basic Research (08-04-00334-

а), and Russian Ministry of Education and Sciences (02.740.11.0079, NS-3185.2010.4).

59

Nitro Derivatives of N-Alkylbenzoaza-18-crown-6 Ethers:

Synthesis and Complexation with Metal and Ammonium Cations

Dmitrieva S.N.1, Churakova M.V.1, Kurchavov N.A.1, Vedernikov A.I. 1,

Freidzon A.Ya.1, Basok S.S.2, Bagatur’yants A.A.1, Gromov S.P.1

1 Photochemistry Center, Russian Academy of Sciences, ul. Novatorov 7A-1,

Moscow 119421, Russian Federation 2 A. V. Bogatsky Physicochemical Institute, National Academy of Sciences of Ukraine,

Odessa 65080, Ukraine

A series of N-alkyl(nitrobenzo)aza-18-crown-6 ethers 1 with the macrocycle nitrogen

atom conjugated with the benzene ring is synthesized. The synthesis is based on stepwise

transformation of the macrocycle of easily available nitrobenzo-18-crown-6 ether 2. The

complexation properties of these compounds are compared with those of the nitro derivatives

of benzo- and N-phenylaza-18-crown-6 ethers using 1H NMR spectroscopy and quantum-

chemical DFT calculations.

O2N OO

O

OO

O

O2N OO

O

NO

OR

O2N OO

O

NO

ORMn+

Mn+

R = Me, Et, n-Pr, CH2Ph

Mn+= Li+, Na+, K+, Ca2+, Ba2+, NH4+, EtNH3

+

12

The stability constants for the crown ether complexes with NH4+, EtNH3

+, Li+, Na+,

and K+ are determined using 1H NMR titration in MeCN-d3. The complexation capacity of

N-alkyl(nitrobenzo)aza-18-crown-6 ethers is much higher than the complexation capacity of

N-(4-nitrophenyl)aza-18-crown-6 ether and is comparable to (or higher than) the similar

characteristics of nitrobenzo-18-crown-6 ether.

This study was supported by the Russian Foundation for Basic Research and the

Russian Academy of Sciences.

60

Effect of acyl moieties in polyethyleneimine backbone on transfection

efficiency and toxicity of the polymer

Efremov A., Rogoza A., Pyshnyi D., Serikov R., Zenkova M.

Institute of Chemical Biology and Fundamental Medicine SB RAS, 8, Lavrentieva ave., 630090, Novosibirsk, Russian Federation

e-mail: efremovlxndr@rambler.ru

Safe and efficient delivery of genetic material remains the most challenging aspect of

human gene therapy. Gene carriers have to overcome multiple extra- and intracellular

obstacles to reach the cell target, including passage across the plasma membrane, escape from

acidic conditions of endocytic vesicles, migration toward the target and release of genetic

material at the appropriate point in this pathway.

Polyethylenimine (PEI) is a promising delivery agent, because of its high buffering

capacity and nucleic acid (NA) condensation ability. Recent studies shown that commercial

PEI 25 kDa, “Polyscience” (lPEI-25) includes up to 11% propionic moieties in the polymeric

backbone. This imperfection in the polymer structure reduces the number of primary amines,

thereby influenced on positive charge and buffering capacity of PEI molecules.

In this research work, we compared cytotoxicity and transfection efficiency of

commercial PEI 25 kDa (lPEI-25) and entirely deacylated PEI with similar molecular weight

(dlPEI-22). Both PEIs show the similar cytotoxic effect on four mammalian cell lines.

Delivery efficiency of NA by PEIs under the study was tested with different N/P ratio, which

is the ratio of the numbers of secondary aminogroups it the polymer to numbers of

internucleotide phosphates of NA. We found that dlPEI-22 is 1.5 times more efficiently

deliver 25-mer fluorescien labeled oligodeoxyribonucleotide into the cells, than lPEI-25,

when amount of FITC-positive cell was measured, while level of average green fluorescent

signal from cell population was similar for both PEIs. Thus, the entire deacylation of lPEI-25

does not lead to significant increase in delivery efficiency, while increasing its price.

This work was supported by grants from RFBR 08-04-00753 a, SS-3689.2008.4, FCP

No. 02.512.11.2200, FCP No. P 438.

61

Recent advances in the field of polyoxothiomolybdenum cycles: Synthesis,

characterization and electrocatalytic properties.

Floquet S. a, Hijazi A. a, Duval S. a, Simonnet-Jégat C. a, Ngo Biboum R. b, Keita B. b,

Nadjo L. b, Haouas M. a, Taulellea F. and Cadot E.a

a Institut Lavoisier de Versailles, UMR 8180, Université de Versailles, 45 av. des Etats Unis, 78035 Versailles, France. sebastien.floquet@chimie.uvsq.fr, cadot@chimie.uvsq.fr

b Laboratoire de Chimie Physique, UMR 8000, Université de Paris XI, Orsay, France

In the last decade, we have derived the first members of a new family of polyoxometalates

(POM) compounds, the cyclic polyoxothiomolybdates, and started exploring their

properties[1-6]. The tremendous propensity of the Mo-rings to encapsulate polycarboxylate

ions led us to use number of polycarboxylate anions and coordination complexes as guest

components. In the present contribution we present some of our recent results obtained in this

field with a particular attention to NMR studies in solution[6,7] and electrochemical

properties of these compounds towards the reduction of protons into hydrogen.[6,8]

1. E. Cadot, B. Salignac, S.Halut, F.Sécheresse, Angew. Chem., Int. Ed., 1998, 37(5), 611. 2. B. Salignac, S. Riedel, A. Dolbecq, F. Sécheresse, E. Cadot, J. Am. Chem. Soc., 2000, 122, 10381. 3. J.-F. Lemonnier, S. Floquet, J. Marrot, E. Terazzi, C. Piguet, P. Lesot, A. Pinto and E. Cadot, Chem. Eur. J., 2007, 13, 3548. 4. A. Kachmar, S. Floquet, J.-F. Lemonnier, E. Cadot, M.-M. Rohmer, M. Bénard, Inorg. Chem., 2009, 48, 6852-6859. 5. J.-F. Lemonnier, S. Floquet, J. Marrot, E. Cadot, Eur. J. Inorg. Chem., 2009, 5233-5239. 6. S. Duval, S. Floquet, C. Simonnet-Jégat, J. Marrot, R. Ngo Biboum, B. Keita, L. Nadjo, M. Haouas, S. Brun, F. Taulelle and E. Cadot, J. Am. Chem. Soc., 2010, 132, 2069-2077. 7. S. Floquet, S. Brun, J.-F. Lemonnier, M. Henry, M.-A. Delsuc, Y. Prigent, E. Cadot and F. Taulelle, J. Am. Chem. Soc., 2009, 131, 17254-17259. 8. B. Keita, S. Floquet, J.-F. Lemonnier, E. Cadot, A. Kachmar, M. Bénard, M.-M. Rohmer, L. Nadjo, J. Phys. Chem. C, 2008, 112, 1109-1114.

62

Novel Cyanine Dyes and Their Self-Assembled Light-Sensitive

Supramolecular Systems

Fomina M. V.1, Nikiforov A. S.1, Vedernikov A. I.1, Kuz’mina L. G.2, Alfimov M. V.1,

Gromov S. P.1

1 Photochemistry Center of the RAS, 7A-1 ul. Novatorov, Moscow 119421, Russian

Federation, e-mail: fomina@photonics.ru 2 N. S. Kurnakov Institute of General and Inorganic Chemistry of the RAS,

31 Leninskiy prosp., Moscow 119991, Russian Federation

Self-assembled light-sensitive supramolecular systems formed by non-covalent

interactions attract considerable attention.

We synthesized new cyanine dyes of the benzothiazole and indolenine series

containing different substituents at the nitrogen atoms.

N

YY

NR R

n

X

+

Y = S, CMe2; n = 1 - 3

The structures of the synthesized dyes were studied by NMR, UV-Vis, IR

spectroscopy and X-ray diffraction.

The synthesized dyes and macroheterocyclic compounds (cavitands) form various

inclusion complexes in aqueous and nonaqeous solutions.

The synthesized cyanine dyes and their supramolecular complexes might be used as

fluorescent probes in biology and in light-sensitive molecular devices.

This work was supported by the Russian Foundation for Basic Research and by the Presidium

of the Russian Academy of Sciences.

63

Binding of human ribosomal proteins S18, S16 and S5 to the major 3’

domain of 18S rRNA

Ilyin A.A., Yanshina D.D., Malygin A.A. and Karpova G.G.

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian

Academy of Sciences, Novosibirsk, 630090, Russia

Understanding processes mediated by the eukaryotic ribosome requires knowledge on its

RNA-protein arrangement, which could not be studied with the use of X-ray crystallography

as it has been done with prokaryotic ribosomes since crystals of eukaryotic ribosomes suitable

for such analysis are not yet available. Nowadays, one of the most applicable approaches to

study RNA-protein interactions in the eukaryotic ribosomal subunits is a method based on

chemical and enzymatic probing of complexes assembled from particular recombinant

ribosomal proteins and RNA transcripts. These complexes simulate various domains of

ribosomal subunits and their structure can be studied using a broad set of probes.

Ribosomal proteins S18, S16, and S5 are the components of the head of human 40S

ribosomal subunit. These proteins are homologous to prokaryotic proteins S13, S9 and S7,

respectively, which together with other proteins bind to the major 3’ domain of 16S rRNA

during the 30S subunit assembly. We have studied binding of recombinant ribosomal proteins

S18, S16, and S5 with an rRNA transcript 3Dm corresponding to the region of human 18S

rRNA containing helices Н28-31 and Н41-43, which is homologous to the region in 16S

rRNA containing the entire binding sites for S7 and S13 and the major part of the site for S9.

Using chemical and enzymatic footprinting, we showed that these proteins protect 3Dm

against hydrolysis with probes mainly in the regions homologous to the sites of their

prokaryotic homologues binding on the 16S rRNA. At the same time, we have found several

regions that do not match to the binding sites the prokaryotic proteins. Comparison of data on

3Dm footprinting in complexes containing one or several ribosomal proteins simultaneously

revealed that each protein affects binding of other ones that may suggest a cooperative effect

of the protein binding on the assembly of the head part of the eukaryotic 40S ribosomal

subunit.

This study was supported by RFBR grant # 08-04-00593-а to A.M.

64

Mapping binding sites of human ribosomal proteins S13 and S16 on RNAs

of various kinds: 18S rRNA and pre-mRNA

Ivanov A. V., Malygin A. A. and Karpova G. G.

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian

Academy of Sciences, Novosibirsk, Russia

Main property of ribosomal proteins is their specifically interaction with different types

of RNAs. During ribosome assembly ribosomal proteins bind to specific sites of rRNA to

form its tertiary structure; in the course of translation, they interact with mRNA as well. In

addition, some ribosomal proteins may bind to their mRNAs and pre-mRNAs and thereby

inhibit own synthesis. In the present study the binding sites of rpS13 on the 18S rRNA and of

rpS16 on its own pre-mRNA were determined by chemical and enzymatic footprnting.

Results obtained by enzymatic footprnting with rpS16 pre-mRNA in its complexes with the

protein showed that major binding site of rpS16 on its pre-mRNA arrange nucleotides of the

first intron nearby the branch point where binding site of snRNP U2 is known to be disposed.

Thus, one can conclude that rpS16 competes with snRNP U2 for binding with the pre-mRNA.

Mapping binding sites rpS13 on the 18S rRNA was carried out using RNA-transcript

corresponding to the central domain of 18S rRNA and a set of rpS13 mutants. Single

mutations of conserved amino acids H101A and D108A and deletions of 27 or 29 amino acids

with N- or C- terminus, respectively, of rpS13 decreased affinity of this protein to the rRNA.

Single mutation Q101A did not affect the binding, whereas deletions of 80 or 54 amino acids

with N- or C- terminus, respectively, of rpS13 abolished binding of the protein to the rRNA.

Chemical and enzymatic footprnting of the rRNA complexed with rpS13 and its mutants

revealed several sites in the 18S rRNA where protection effects caused by the bound protein

were observed. These sites include nucleotides G680 и U682 (helix 20), A926, G936 and

A938 (helix 22), C985 and C989 (helix 23), A1033 and A1050 (helix 24) и C1096 и A1101

(helix 26). Deletion of 27 N-terminal amino acids decreased extent of protection of

nucleotides in helixes H20 and H24 whereas lack of 29 C-terminal amino acids abrogated

protection of nucleotides in helixes H22 and H26. The data obtained allow conclude that

human rpS13 have additional binding site on the 18S rRNA (helix 24) as compare to the

prokaryotic ribosome. This helix contacts with eukaryotic specific N-terminus of rpS13.

This work was supported by grants from the Russian Foundation for Basic Research (08-04-00593-a) to A.M. and by the Russian Academy of Sciences Presidium program ‘‘Molecular and cell biology’’ to G.K.

65

ReO4-, a weak ligand of U(VI): a complexation and extraction study in ILs

Klimchuk O.a, c, Ouadi A.a, Georg S.a, Billard I.a, Gaillard C.b

a IPHC, DRS/CHNU, 23 rue du Loess, 67037 Strasbourg Cedex 2, France

b Institut de Physique Nucléaire de Lyon, 4 rue Enrico Fermi, 69622 Villeurbanne, France c Institute of Organic Chemistry National Academy of Sciences of Ukraine, Murmanska str.,

502660, Kyiv-94, Ukraine

Room Temperature Ionic Liquids (RTILs) are a new class of “green” solvents. Their

physico-chemical properties are easily tunable by changes in their chemical structure, so that

they are often called “design solvent”. Although such aspects are indeed rather interesting in

view of potential industrial applications (ILs are, most of the time, non flammable and non

volatile, but this rule suffers from exceptions), to our opinion, the unusual solvation properties

of these media are a much more appealing advantage.

We have investigated the solvation, complexation and extraction of UO22+ towards

ReO4- that are of industrial relevance to the nuclear fuel cycle, in three different hydrophobic

ILs (tributylmethylammoniumTf2N, trimethylbutylammoniumTf2N and 1-methyl-3-

butylimidazoliumTf2N, where Tf2N– stands for (CF3SO2)2N-). Experiments in water have

been performed for comparison purposes. Complexation and extraction studies in ILs have

implied TBP (tributylphosphate), a well-known extractant of such ions.

The results of complexation and extraction will be presented.

Ionic liquids are shown to be media in which an order of complexing strength for weakly

coordinating anions, which can not be obtained in water, can be derived.

66

Sorption of catalytically active species into mesoporous coordination

polymer MIL-101 Kovalenko K.A.1, Maksimchuk N.V.2, Kholdeeva O.A.2, Fedin V.P.1

1 Nikolaev Institute of Inorganic Chemistry SB RAS, Novosibirsk, Russia 2 Boreskov Institute of Catalysis SB RAS, Novosibirsk, Russia

e-mail: k.a.kovalenko@niic.nsc.ru

Today coordination polymers and metal-organic frameworks (MOFs) have a great

attention in inorganic and supramolecular chemistry due to very promising applications,

especially in gas sorption and separation, producing of new hybrid materials and catalysis.

One of the most outstanding MOF was developed by prof. G. Férey in 2005 [1]. In

hydrothermal reaction between chromium nitrate and terephthalic acid in aques solution with

equal amount of HF as mineralizing agent the mesoporous chromium terephthalate

Cr3O(H2O)2Fx(NO3)1−x (O2CC6H4CO2) · nH2O (n ≈ 15–20), also called MIL-101, was

produced. MIL-101 possess unprecedented large surface area (up to 5900 m2/g) and pore size

(3.4 nm and 3.8 nm). Furthermore this material is very thermal (up to 300 °C) and

hydrolitically stable. All these features make MIL-101 a good candidate as support for

different catalytically active species such as polyoxometallates (POMs) [2, 3].

We was investigated the sorption of Keggin POMs (PW12O403– and SiW12O40

3–) on MIL-101.

Due to positive charged MIL-101 matrix and anionic character of guest the anionic exchanged

mechanism of sorption was supposed and proved by measurements of POMs concentrations

in solution after sorption and amount of absorbed POMs on MIL-101, and also residual nitrate

anions leaching from MIL-101 was measured. POMs interact with surface of MIL-101 very

strong and 5–15% of POMs not desorb even after washing by distiller water several times.

1. G. Férey, C. Mellot-Draznieks, C. Serre, F. Millange, J. Dutour, S. Surblé, I. Margiolaki.

Science, 2005, 309, 2040.

2. N.V. Maksimchuk, M.N. Timofeeva, M.S. Melgunov, A.N. Shmakov, Yu.A. Chesalov,

D.N. Dybtsev, V.P. Fedin, O.A. Kholdeeva, J. Catal., 2008, 257, 315.

3. N.V. Maksimchuk, K.A. Kovalenko, S.S. Arzumanov, Y.A. Chesalov, M.S. Melgunov,

A.G. Stepanov, V.P. Fedin, and O.A. Kholdeeva. Inorg. Chem., 2010, 49, 2920.

This work was supported by the Russian Foundation for Basic Research (09-03-90414, 09-

0312112). K.A. Kovalenko also thanks the Haldor Topsøe A/S for Ph.D. Scholarship.

67

Application of selective probes based on nitroxides attached to cyclodextrin

(CD) for fluorescence quenching

Krumkacheva O.1, Fedin M.1, Polovyanenko D.1, Marque S.2, Plyusnin V.F.3 and

Bagryanskaya E.1

1 International Tomography Center SB RAS, Novosibirsk, Institutskaya 3A,Russia 2Aix-Marseille Université, UMR 6264-LCP,13397 Marseille Cedex 20, France 3 Institute of Chemical Kinetics and Combustion SB RAS, Novosibrisk, Russia

Similar to many spectroscopic techniques, the main drawback of fluorescence

spectroscopy is the overlap of lines of various fluorophores that complicates the analysis of

data. One possible solution of this problem is the application of efficient and selective

quenching agents leading to enhancement of the contrast. In this work we investigated the

quenching agent based on nitroxides covalently attached to cyclodextrines.

Processes of self-inclusion of nitroxides into the

cavities were studied by means of cw-EPR, Electron

Spin Echo Envelope Modulation (ESEEM) and NMR

techniques. Using 1-adamantanol as a competitor, we

have shown that the capping nitroxide is favored over

the deeply-included nitroxide. This result was nicely confirmed by the absence of changes in

cw-EPR signal and striking changes in ESEEM using free and attached nitroxides. The

efficiency of the fluorescence quenching of methyl orange and other fluorophores was

investigated using the fluorescent technique. We show that the concept of forced electron-

chromophore interactions is strikingly efficient to quench the fluorescence of guest molecules,

and that the use of recognition properties of CD and bleaching properties of nitroxides allow

for efficient and selective quenching agent.

The financial support by grant of the RFBR 08-04-00555 and Russian Federal Agency for

Education project N 1144.

N

C

LN

O

X

N

O

X

N

NH2N

O

1 2 3 X = OH, NH2

N

O

NH

O

O

NH

NHL

cyclodextrinC

1 2

methyl-(full methylation of hydroxyl groups on the rims)

68

Investigation of Electron Structure of calixarenes thioethers and

thiacalixarenes by XPS, XES and quantum chemical methods.

Kryuchkova N.A.1, Mazalov L.N.1, Fedorenko E.V.1, Korotaev E.V.1, Torgov V.G.1,

Kostin G.A.1, Kalchenko V.I.2

1 Institute of Inorganic Chemistry SB RAS, Novosibirsk, Russia

2 Institute of Organic Chemistry NAN Ukraina, Kiev, Ukraina

Sulfur-containing calixarenes are of great interest for making macrocyclic receptors

for noble metals (Au, Ag, Pd). Due to chelate effect traditional calixarenes grafted with

thioether groups provide better metal capture compared with monodentate analogues. Change

of CH2-bridging fragments in calixarenes to sulfur atoms in thiacalixarenes provide additional

donor centers also affecting on geometry and electron structure of molecule in whole.

The complex analysis of the upper rim grafted calixarene thioethers and

thiacalixarenes have been carried out (XPS, XES and quantum chemical investigation). The

X-ray photoelectron spectra (S2p3/2, O1s, P2p3/2, Pd3d5/2 and Cl2p3/2) were recorded on a VG

ESCA-3 electronic spectrometer using nonmonochromated AlKα radiation (h = 1486.6 eV).

We also investigated the SKα and SKβ X-ray emission spectra of sulfur atoms. All the

quantum-chemical calculations were carried out with Jaguar 6.5 packages using density

functional theory and B3LYP hybrid functional (basis M6-31G+*(TM)). The theoretical

model spectra were constructed based on quantum chemical calculation. These model spectra

allow to define the binding energies of HOMO and quality

Summary of K(S) and XPS spectra shown that charge state of sulfur atoms in

thiacalixarenes and calixarene thioesters is approximately the same. In palladium complexes,

the electron density on sulfur atom decrease because of the coordination to Pd. The analysis

of molecular orbitals and sulfur K spectra shown the absence of -interactions between

sulfur atoms and arene rings in contrast to diphenyl sulfide.

69

Benzothiacrown ethers: synthesis and structure of their complexes with

palladium(II) Kurchavov N.A.1, Dmitrieva S.N.1, Sidorenko N.I.1, Vedernikov A.I.1, Freidzon A.Ya.1,

Bagatur’yants A.A.1, Kuz’mina L.G.2, Gromov S.P.1

1 Photochemistry center, Russian Academy of Sciences, Novatorov-7A-1, Moscow 119421,

Russia 2 N.S. Kurnakov Institute of General and Inorganic Chemistry, Russian Academy of Sciences,

Leninskiy pr. 31, Moscow 119991, Russia

A synthesis of benzodithiacrown ethers (L) with various-sized macrocycles is

proposed. The synthesized macrocycles form complexes of the formula [PdLCl2] and

[PdL(OAc)2] with [Pd(MeCN)2Cl2] and Pd(OAc)2 salts.

S

OO

SOX

n

S

OO

SOX

PdA2

n

EtOH - H2O

YO

YOX

SH

O

SH

n M2CO3

X = NO2, CHO; Y = Cl, I; n = 0 - 3; M = Li, Na, K, Cs; A = Cl, OAc

[Pd(MeCN)2Cl2] or Pd(OAc)2

+

The structure of the benzothiacrown ethers and their complexes with palladium (II)

salts is studied by 1D and 2D NMR methods. The electronic and geometrical structures of the

free crown ethers and their complexes with palladium (II) chloride are calculated by DFT.

The calculated structures are used for the interpretation of the NMR data.

The structures of free benzothiacrown ethers and their complexes with PdCl2 are

studied by X-ray diffraction. It is found that palladium(II) is bound to the two sulfur atoms of

the macrocycle and two chloride anions and lies in the center of a square S2PdCl2. In 12- and

18-membered macrocycles, the sulfur atoms in the S2PdCl2 fragment are in the cis position. In

21-membered macrocycles, the sulfur atoms in the S2PdCl2 fragment are in the trans position.

These structure data agree with the NMR and theoretical data.

This work was supported by Russian Foundation for Basic Research and Russian

Academy of Sciences.

70

PARP2 binds to and is activated by DNA structures mimicking DNA repair

intermediates Kutuzov M. M.1, Ame J.-C.2, Khodyreva S. N.1, Schreiber V.2, Lavrik. O. I.1

1Institute of Chemical Biology and Fundamental Medicine, Novosibirsk, Russia, 2FRE3211, IREBS, CNRS, Université de Strasbourg, ESBS, France

Poly(ADP-ribosyl)ation, one of the posttranslational protein modifications significant

for genomic stability and cell survival in response to DNA damage, is mostly catalyzed by

PARP1. It is the first protein described to synthesize PAR in response to mitogenic stimuli or

genotoxic stress. PARPs now constitute a large family of 17 proteins displaying a conserved

catalytic domain, in which PARP1 and PARP2 are so far the sole enzymes whose catalytic

activity is immediately stimulated by DNA strand-breaks. But DNA-binding domain of

PARP2, in contrast to PARP1 containing Zn-fingers, based on SAP-motif that potentially

suggests differences in the specificity of DNA recognition and allows to hypothesize different

roles of these enzymes. Whereas the contribution of PARP1 in the response to DNA damage

has been widely illustrated, the contribution of the other DNA dependent PARP, PARP2, has

not been studied so far and the most part of published data based on the experiments on cells

or on mice. There are some data about requirements of PARP2 for efficient base excision

repair, homologous recombination, DNA stability during mitosis and differentiation of post-

meiotic germ cells. From the other hand, target of PARP2, its role and the mechanism of

involvement to different DNA-dependent processes are still not very clear.

In this work few DNA-structures (nick-, gap2-, gap10-, gap20-, flap3-, flap9-, over5-,

over3-, hp-, twj-, fwj-DNAs) mimicking intermediates of different DNA-metabolism

processes were used to identify the structure which PARP2 could be addressed to. To

determine the contribution of PARP1 and PARP2 to the total poly(ADP-ribose) synthesis the

comparison of activation was performed with each of DNA. For that activation of PARP1/2

by these DNAs and the Kd of PARP2 were measured. As expected, the activity of PARP2 was

much lower than PARP1's activity in presence of the same DNAs. Surprising, that for PARP2

the correlation between activation efficiency and Kd wasn't found. PARP2 was activated the

most effectively in the presence of over5- and gap2-DNAs but it displayed higher affinity to

gap20- and flap9-DNAs. Taken together this data allow to count effectivity of poly(ADP-

ribose) synthesis by PARP2 as Vmax/Kd and to conclude that over5- and gap20-DNAs are the

most effective activators for PARP2. Acknowledgements: This work was supported by RFBR, project № 10-04-01083, Program of RAS

“Molecular and Cellular Biology”.

71

Phthalocyanine-oligonucleotide conjugates for DNA oxidation by O2 and

H2O2

Kuznetsova A.1,2, Solovieva L.3, Lukyanets E.3, Kaliya O.3, Knorre D.2, Fedorova O.1,2

1 Novosibirsk State University, Novosibirsk, Russia; 2 Institute of Chemical Biology and Fundamental Medicine, Siberian Division of Russian

Academy of Sciences, Novosibirsk, Russia; 3 State Scientific Centre “NIOPIK”, Moscow, Russia

Some metal-phthalocyanine complexes are intensively used over the last decades

in the photodynamic and catalytic therapies of cancer. The antitumor action of these

complexes is connected with the production of ROS.

The present work was aimed at the investigation of single-stranded oxidation of

DNA by O2 and H2O2 in complementary complexes with Co(II)- or Fe(II)-phthalocyanine-

oligonucleotide conjugates. In addition, the ssDNA oxidation by O2 and H2O2 in the presence

of dimeric complexes of negatively and positively charged Fe(II)- and Co(II)-phthalocyanines

(MePcpos•MePcneg) was investigated. These complexes were formed directly on ssDNA due to

binding of negatively charged phthalocyanine MePcneg in conjugate with free positively

charged Co(II)- or Fe(II)-phthalocyanine (MePcpos).

We have shown that ssDNA is efficiently damaged in the presence of H2O2 and

complementary conjugate. The dimeric complexes MePcpos•MePcneg showed significant

increase of catalytic activity compared with MePcneg. MePcpos•MePcneg catalyzed the DNA

oxidation by O2 with high efficacy and led to direct DNA strand cleavage. In all cases the

guanine residues located close to the source of the oxidizing species in the complementary

complex were the most susceptible to modification.

We investigated the dGMP oxidation as a model compound to provide molecular

insight into the structure of guanine lesions generated by MePc. The dGMP oxidation by O2

catalyzed by MePc led to formation of 1,N2-glyoxal-adduct of dGMP and 7,8-dihydro-8-oxo-

dGMP. The formation of 8-oxoguanine derivatives, as well as the products of sugar oxidation,

could be evidence of several oxidizing species in the reaction mixture: hydroxyl radicals and

high valent metal-oxo species. Our results show that MePc as a reactive group within

oligonucleotide conjugates are very effective towards DNA oxidation by O2 and H2O2.

Supported by a Grant from RFBR (08-04-00334-а), President Grant (NS-

3185.2010.4), Grants from Russian Ministry of Education and Sciences (2.1.1/1499 and

02.740.11.0079).

72

Mechanism of DNA repair by human 8-oxoguanine-DNA-glycosylase

hOgg1 Kuznetsov N.1, Koval V.1,2 and Fedorova O.1,2

1Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk 630090, Russia; 2Novosibirsk State University, Novosibirsk, Russia

Cellular DNA is continuously attacked by reactive oxygen species leading to many

lesions. One of the most common lesions is 7,8-dihydro-8-oxoguanine (8-oxoG). In human

cells, the effects of 8-oxoG are counteracted by hOgg1, a DNA glycosylase that catalyzes

excision of 8-oxoG base followed by a much slower β-elimination reaction at the 3′-side of

the resulting abasic site. Many features of hOgg1 mechanism, including its low β-elimination

activity and high specificity for a cytosine base opposite the lesion, remain poorly explained

despite the availability of structural information. In this work, we have analyzed the substrate

specificity and the catalytic mechanism of hOgg1 acting on various DNA substrates using

stopped-flow kinetics with fluorescence detection. Combining data on intrinsic tryptophan

fluorescence to detect conformational transitions in the enzyme molecule and 2-aminopurine

reporter fluorescence to follow DNA dynamics, we have defined three pre-excision steps and

assigned them to the processes of (i) initial encounter with eversion of the damaged base, (ii)

insertion of several enzyme’s residues into DNA, and (iii) enzyme isomerization to the

catalytically competent form [1]. The individual rate constants were derived for all reaction

stages. Of all conformational changes, we have identified the insertion step as mostly

responsible for the opposite-base specificity of hOgg1 towards 8-oxoG:C as compared with 8-

oxoG:T, 8-oxoG:G, and 8-oxoG:A. We have also investigated the kinetic mechanism of

hOgg1 stimulation by 8-bromoguanine and showed that this compound affects the rate of β-

elimination rather than pre-excision dynamics of DNA and the enzyme [2].

1. Kuznetsov N.A., Koval V.V., Zharkov D.O., Nevinsky G.A., Douglas K.T. and Fedorova

O.S. (2005) Nucleic Acids Res. 33, 3919-3931.

2. Kuznetsov N.A., Koval V.V., Nevinsky G.A., Douglas K.T., Zharkov D.O. and Fedorova

O.S. (2007) J. Biol. Chem. 282, 1029-1038.

The research was supported by grants from the RFBR № 10-04-00070 and 08-04-

12211, the Siberian Division of the Russian Academy of Sciences № 28 and 48, the Russian

Ministry of Education and Science № 02.740.11.5012, NS-3185.2010.4 and MK-

1304.2010.4.

73

Styrylheterocycles for light-sensitive supramolecular systems

Lobova N.A., Vedernikov A.I., Aleksandrova N.A., Iskhakova I.T., Alfimov M.V.

and Gromov S.P.

Photochemistry Center of the RAS, 7A Novatorov str., Moscow 119421, Russia

The synthesis of novel styrylheterocycle derivatives of pyridine, quinoline and

benzothiazole series with (and without) N-substituents was developed. Two main approaches

were used: condensation of formylarylcrown ether with suitable heterocyclic quaternary salt

or quaternization of styrylheterocycle. Styrylheterocycles having no crown-ether fragment

were used as reference compounds.

N N

S

N,, =

OO

O

OO

O

m

N

OO

O O

Om

m = 0, 1

= ,

RR

= cavitand

The formation of inclusion complexes between styrylheterocycles and cyclodextrins

and cucurbit[n]urils was studied using different spectral methods. The stability of complexes

depends on styrylheterocycle substituents nature and host cavity size and type.

This work was supported by the Russian Foundation for Basic Research and

Presidium of the Russian Academy of Sciences.

74

Inclusion complexes of functional nitroxides with cucurbit[7]uril

Polovyanenko D.1, Kirilyuk I. 2, Semenov S.1

Grigor'ev I.2, Geras’ko O.3, Fedin V.3 and Bagryanskaya E.1

1 International Tomography Center SB RAS Novosibirsk, 630090, Russia

2 Novosibirsk Institute of Organic Chemistry SB RAS, Novosibirsk, 630090, Russia 3 Nikolaev Institute of Inorganic Chemistry SB RAS, Novosibirsk, 630090, Russia

E-mail: polo@tomo.nsc.ru

Nitroxide radicals are widely used paramagnetic probes for measurement of pH, redox

status, oximetry, nitric oxide detection using electron paramagnetic resonance (EPR)

spectroscopy. The main problem of nitroxides wide application is a fast reduction to EPR

silent diamagnetic hydroxylamines. Inclusion of nitroxides into the cavity of molecular

nanocontainers (such as cyclodextrins, calixarenes and cucurbiturils) can significantly retard

the reaction of nitroxides with reductants and increase nitroxide lifetime.

In this work possible application of cucurbit[7]urils as a nanocontainers for pH-sensitive

nitroxides was studied. Inclusion complexes of nitroxides of pyrrolidine and imidazoline

series with cucurbit[7]uril (CB7) were studied using X-band EPR and NMR methods.

It was found that reversible formation of inclusion complexes of nitroxides is

accompanied by a decrease of hyperfine interaction (HFI) constant on nitrogen of the

nitroxide group and by a 5-7-fold increase of rotational correlation time monitored by EPR.

The binding constants of the nitroxides and CB7 were determined. The influence of alkali

metal ions and pH on the equilibrium free - encapsulated nitroxides and binding constants was

studied. The EPR spectra of CB7 mixtures with nitroxides were found to be more sensitive to

pH changes than the spectra of pure nitroxides. Apparent pK of these mixtures was found to

increase with CB7 concentration. The nitroxide@CB7 complexes showed higher resistance to

chemical reduction with ascorbic acid compared to free nitroxides. Thus, cucurbit[7]uril can

be used for the improvement of functional properties and stability of nitroxide radicals.

The financial support by grant of the RFBR 08-04-00555 and Russian Federal Agency

for Education project N 1144.

References

1. Kirilyuk I.A., Polovyanenko D. N. et al. J.Phys. Chem. B. 114 (2010) 1719-1728.

75

Structural and Calorimetric Studies of Ionic Clathrate Hydrates of

Tetrabutylammonium Chloride Rodionova T., Manakov A., Komarov V., Villevald G., Karpova T.

Nikolaev Institute of Inorganic Chemistry SB RAS 3, Acad. Lavrentieva avenue, Novosibirsk, 630090

In crystal structures of the ionic clathrate hydrates of tetrabutylammonium halogenides water molecules together with halogenide anions form the polyhedral water - anion host framework through hydrogen-bonding. Butyl groups of cations are incorporated in polyhedral cavities of water-anion framework so that distances between cation atoms and water framework are no less than a sum of their van der Waals radii. The ionic clathrate hydrates of tetraalkylammonium salts are extensively investigated in the last decade as potentially available for manifold applications. Owing to existing of empty cavities in the crystal structures they can be used for storage, transportation and separation of gases, as well for storage and transportation of cold. In spite of considerable number of investigations of these compounds their crystal structures are still unknown and thermodynamic data are controversial in many cases. The object of the present work is to study structures of ionic clathrate hydrates of tetrabutylammonium chloride and their enthalpies of fusion.

Previously studied Т,Х phase diagram of (C4H9)4NCl – H2O binary system and isotherms of (C4H9)4NCl – H2O – NH4Cl ternary systems showed that there are three ionic clathrate hydrates in clathrate formation field. We synthesized these polyhydrates, determined their stoichiometry and enthalpies of fusion: (n-C4H9)4NCl.32.20.4 H2O, Hf = 4.9 kJ/mol hydrate (5.56 kJ/mol H2O); (n-C4H9)4NCl. 29.70.4H2O, Hf = 156.9.1 kJ/mol hydrate (5.28 kJ/mol H2O); (n-C4H9)4NCl. (24.80.3)H2O, Hf = 127.9.6 kJ/mol hydrate (5.16 kJ/mol H2O). The stoichiometry was determined by measuring of concentration of tetrabutylammonium cation by potentiometric titration with a sodium tetraphenylborate solution and an ion-selective electrode. The differential scanning calorimeter DSC-111 (Setaram) was used for determination of enthalpies of fusion.

Single crystal X-ray analysis has been performed at 150 K. Four X-Ray data collection experiments were carried out. According to unit cell metrics and peculiarities of data sets they can be divided in two structure types. Both types are solved in P42/m. Idealized host water frameworks correspond to tetragonal structure-I (TS-I). Distinctions between two types of structures result from various inclusion modes of guest entities. New manner of halogenide anion inclusion with replacing of two host water molecules has been revealed. The possibility of the formation of various structures of polyhydrates on base of the TS-I host water framework has been shown. The results of X-ray powder diffraction studies of polyhydrate samples supported this conclusion.

76

New intergrowth structure type of ionic clathrate hydrates Komarov V.Yu., Rodionova T.V., Solodovnikov S.F., Kuratieva N.V.

Nikolaev Institute of Inorganic Chemistry SB RAS, Acad. Lavrentiev Ave. 3, Novosibirsk, 630090

Ionic clathrate hydrates are classical objects belonging to the field of inclusion phenomena

chemistry. Host frameworks of these compounds are built of hydrogen-bonded water molecules

and ions; included counterions are regarded as guest species. At the present time ionic clathrate

hydrates of tetra-n-butyl- (TBA) and tetra-iso-amylammonium (TiAA) salts are considered as

promising materials for storage and transport of energy carriers (e.g., hydrogen), energy-

saving («cold storage»), and development of gas mixture separation technologies. Tailoring of

these materials requires, in particular, understanding of their structural chemistry.

XRD investigation of two crystals grown from water solutions of TiAA X (X– = Br–, I–)

are presented in this work. Structural models obtained are similar in both cases (space group

Fmm2, approximate unit cell dimensions a = 21.7 Å, b = 47.6 Å and c = 11.9 Å) and have

TiAA X·35H2O stoichiometry, the host framework being unprecedented.

… (D3)n (T2P2)n (D3)n … … (D3)n (TP3)n (d2)n (TP3)n (D3)n (TP3)n (d2)n … … (d2)n (P4)n (d2)n …

The idealized host framework of these compounds has the highest symmetry Fmmm and

may be described as a close packing of polyhedral cages of four types: d (4258, containing two

quadrilateral and eight pentagonal faces), D (512), T (51262) и P (51263). This tetrahedral

framework with 8d·12D·8T·24P·296H2O unit cell content may be described as a hybrid (see

picture) of two frameworks known early (HS I, 2P·2T·3D·40H2O and TS II, 8P·4d·68H2O)

which are different in arrangement of layers of P cavities. In the structural chemistry of

clathrate hydrates, the structures studied are the first examples of intergrowth phases which

are well-known among layered perovskites. Perhaps, these clathrate hydrates could be

members of homologous series like to Aurivillius and Ruddlesden-Popper perovskite phases.

Financial support from Russian Foundation of Basic Research (Grant 09-03-00367) is gratefully acknowledged.

77

Methane Sorption and Structural Characterization of the Sorption Sites

in Zn2(bdc)2(dabco) by Single Crystal X-ray Crystallography

Samsonenko D.G.1,Dybtsev D.N.1, Berdonosova E.A.2, Hyunuk Kim,3 Kimoon Kim3

1 Nikolaev Institute of Inorganic Chemistry SB RAS, 2 Department of Chemistry, Lomonosov Moscow State University,

3 National Creative Research Initiative Center for Smart Supramolecules, and Department of Chemistry, Pohang University of Science and Technology, Republic of Korea

Sorption isotherms of methane in

Zn2(bdc)2(dabco) were measured up to 35 bar in

a temperature range of 198–296 K. The methane

sorption measurements at 296 K showed an

uptake of 137 cm3/cm3 at 35 bar. The enthalpy

of methane adsorption for Zn2(bdc)2(dabco)

estimated by the virial equation is 13.6 kJ/mol at

zero coverage. X-ray structure analysis of

methane-adsorbed Zn2(bdc)2(dabco) by synchrotron radiation at 90 K revealed that methane

molecules occupy three independent sorption sites (A, B, and C) with a stoichiometry of

Zn2(bdc)2(dabco)·6.69CH4, which is consistent with the results of the gas sorption

measurements at 198 K. In a cavity, eight symmetry-related methane sorption sites A are

located near the {Zn2(CO2)4} paddle-wheel units, while four symmetry-related methane

sorption sites B are near the center of the small windows along the a and b axes. Both A and

B sites are half-occupied. Methane molecules occupying sites A are not only in van der Waals

contacts with the paddle-wheel units, but also interact with the phenyl rings of bdc ligands

through partial π-HC interactions. Methane molecules in B sites interact with the side of the

phenyl rings through van der Waals interaction. The site C, located at the center of the cavity,

is a secondary sorption site; methane molecules occupying sites C are in van der Waals

contact with those in sites A and B.

This study was supported by the Russian Foundation for Basic Research (grants 09-03-90414, 09-03-12112).

78

Synthesis of Microporous Metal-Organic Frameworks and Applications of

their Sorption Properties

Sapchenko S.A.a,b, Samsonenko D.G.a,b, Fedin V.P.a,b

a Nikolaev Institute of Inorganic Chemistry, Siberian Branch of the Russian Academy of Sciences, 3 Akad. Lavrentiev Av., 630090 Novosibirsk, Russian Federation.

b Novosibirsk State University, 2 Pirogova street, 630090 Novosibirsk, Russian Federation

Among the metal-organic frameworks, coordination polymers based on zinc carboxylates

play a special role because of large number of possible building units, varying from Zn(II)

monomers to small di-, tri-, tetranuclear clusters or even infinite chains, assembled into robust

porous frameworks with catalytic, luminescent and gas sorption properties

Herein we report the synthesis, structure, thermal luminescent and sorption properties of

three new microporous metal-organic coordination polymers

(NH2(CH3)2)2[Zn3(bdc)4]·DMF·H2O (1), (NH2(CH3)2)2[Zn3(bpdc)4]·5DMF (2) и

[Zn4(ndc)4(ur)2(dmf)]·DMF (3), (H2bdc = 1,4-benzenedicarboxylic acid, H2bpdc = 4,4'-

biphenyldicarboxylic acid, H2ndc = 2,6- naphthalenedicarboxylic acid, ur = methenamine).

The single-crystal X-ray structure analyses reveal compounds 1 and 2 have 3D anionic

framework structure built from zinc(II) carboxylate layers linked by carboxylate anions.

Compound 3 is a neutral framework (see fig.) with permanent porosity and high surface area.

Guest DMF molecules within channels of 3 (9.5×10 Å) can be evacuated without loss of

sample’s crystallinity. Guest-free material demonstrates adsorption not only of light gases but

also aromatic hydrocarbons such as benzene as well.

Compounds 1-3 also exhibit photoluminescent

properties that can be described in terms of LMCT

mechanism.

This study was supported by the Russian

Foundation for Basic Research (grants 09-03-90414,

09-03-12112). A grant of the Russian Academy of

Sciences (program No. 5.6.1) and a grant of the

Siberian Branch of the Russian Academy of Sciences

(program No. 107) are gratefully acknowledged.

79

Artificial Box C/D RNA as an Instrument of Precise Modification of

Cellular RNA

Semenov D.V., Stepanov G. A., Koval O.A., Kuligina E.V., Richter V.A.

Institute of Chemical Biology and Fundamental Medicine SB RAS, Lavrentiev Ave., 8,

Novosibirsk, 630090, Russia. E-mail: semenov@niboch.nsc.ru

Box C/D small nucleolar RNAs (snoRNA) guide the site-specific 2'-O-ribose

methylation of nucleotides in rRNAs and snRNAs. Recently it has been shown that box C/D

snoRNA and their fragments regulates post-transcription modifications and alternative

splicing of pre-mRNA. In this study we designed and synthesized artificial analogs of U24

snoRNA directed to nucleotides of rRNA and pre-mRNA, and mature mRNA. It was found

that transfection of cultured human cells by artificial snoRNA guiding 2'-O-methylation of

pre-mRNA induced partial splicing impairments. Transfection by artificial snoRNA directed

to rRNA induced de novo 2'-O-methylation of targeted nucleotides of rRNA and can decrease

viability of human cells. These data emphasize the structure of box C/D RNA as a promising

basis for the development of new biologically-active substances.

80

Supramolecular complexes of nucleic acids are potential tools for

siRNA cell delivery

Serikov R., Efremov A., Vlassov V., Zenkova М.

Institute of Chemical Biology and Fundamental Medicine SB RAS 8, Lavrentiev ave., 630090, Novosibirsk, Russian Federation e-mail: serikov@niboch.nsc.ru

A number of gene-targeted therapeutics, e.g. antisense oligonucleotides, siRNAs and

ribozymes, are able to affect intracellular gene expression and to silence genes of infectious

agents or unwanted host cell genes. Poor uptake of nucleic acids by cells and their

degradation due to activity of cellular nucleases remain to be the main obstacle significantly

complicating their research and clinical implementation. Cationic compounds such as cationic

lipids, polyethyleneimine and its derivatives are widely used to improve transfection

efficiency and to prevent nucleic acids digestion by nucleases, the most significant obstacles

for use of these cationic compounds in medicine being their high toxicity and inactivation by

binding with serum proteins.

In this work we investigated a novel system for siRNA delivery into mammalian cells

employing formation of supramolecular complexes by hairpin RNAs of specially designed

structure. These hairpins spontaneously formed the supramolecular complexes 1000 – 2000

b.p. in length by cooperative complementary interactions between 3'-overhangs of the

hairpins and by loop-loop interactions. A 5-nucleotides bulge loop was introduced into the

stem of RNA sequence to govern favorable hybridization kinetics. In the cell these hairpin

RNAs are processed to corresponding siRNA and efficiently silence the target gene.

This work was supported by the grants RFBR 08-04-00753-а, FCP No.

02.512.11.2200, FCP No. P 438, SS-3689.2008.4.

81

Clathrate Hydrates of Polymeric Guest Molecules. Physico-chemical and Structural Studies of Clathrate Hydrates of Tetrabutylammonium and

Tetraisoamylammonium Polyacrylates. Terekhova I.S.1, Manakov A.Yu.1, Soldatov D.V.2, Skiba S. S.1, Stenin Y.G.1,

Villevald G.V.1, Karpova T.D.1

1Nikolaev Institute of Inorganic Chemistry, Siberian Division of RAS, Novosibirsk, Russia 2Department of Chemistry, University of Guelph, Guelph, Ontario, Canada

In this work the results are presented of physico-chemical and structural studies of the

polyhydrates crystallizing at positive temperatures in swelled grains of certain type of carboxylic cationites in the tetraisoamylammonium (TiAA) and tetrabutylammonium (TBA) form – cross-linked TiAA and TBA polyacrylates with low degree of cross-linking.

The study of hydration of synthetic polyelectrolytes, containing carboxylate-ions and tetraalkylammonium counter-ions in the side chains, may be of interest as modelling hydration processes involving biopolymers. The clathrate nature of the studied compounds suggested earlier [1] enables also to consider them as potential reservoirs with molecular-size cavities for gas storage and separation.

The phase diagram studies of the binary water systems with given above polymeric molecules using DTA technique allowed to determine the composition and stability regions of the hydrates formed in the system. X-ray powder diffraction studies were performed of the polyhydrates studied and the type of crystal structure was determined. It was found that polyhydrates of TiAA polyacrylates crystallize in hexagonal symmetry (a=12,25 Å, c= 12,72 Å, 276 K) and TBA polyacrylate polyhydrates – in tetragonal symmetry (a= 23,60 Å, c= 12,41 Å, 248 K) that is characteristic for the clathrate hydrates of tetraalkylammonium salts with simple anions [2]. Powder X-ray diffraction analyses of the hydrate with linear TiAA polyacrylate revealed the identity of the crystal structure with that of cross-linked TiAA polymer. Therefore, to solve the structure of cross-linked TiAA polyacrylate polyhydrate a hydrate with linear polyacrylate was attempted as a first model approxiamtion. Based on the data obtained the structural model for the hydrates with cross-linked polymer was suggested.

In order to evaluate quantitatively the stability of the hydrate framework depending on the degree of cross-linking of the polymeric anion and on the size of the cation the heats of fusion were measured by DSC method for the clathrate hydrates studied.

[1] Bogatyryov, V.L. Ion Exchange; Marcel Dekker: New York. Basel, 1999; Vol.1.

[2] Jeffrey, G.A. Comprehensive Supramolecular Chemistry; Pergamon Press: Oxford,1996; Vol.6.

82

Kinetic mechanism of human AP endonuclease (APE1) in nucleotide

incision repair pathway (NIR). The influence of mutations K98A and ΔN61.

Timofeyeva N. A.1, Koval V. V.1, Ishchenko A. A2, Saparbaev M. K2, Fedorova O. S.1

1 Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, and Novosibirsk State University, Novosibirsk, 630090, Russia.

2 Groupe «Réparation de l’ADN», UMR8200 CNRS, Institut Gustave Roussy, 94805 Villejuif Cedex, France

The repair of certain oxidative base lesions can be initiated by APE1 in nucleotide

incision repair (NIR) pathway, bypassing the glycosylase step and enabling to avoid

formation of a potentially toxic AP-site (AP) intermediate. We analyzed the conformational

dynamics and the kinetic mechanism of APE1 action on 5,6-dihydro-2’-deoxyuridine (DHU)

containing substrate in NIR process in pre-steady-state conditions using a stopped-flow

method. The influence of REF1 domain (N-terminal 61 amino acids of APE1) and of Lys-98

substitution for Ala (APE1K98A) on kinetic parameters of APE1 action has been studied. Our

results permit to propose that APE1 had at least three centers for DHU-substrate binding.

Almost all APE1 molecules possessed a binding activity. Using [32P]-labelled DHU-substrate

we have shown, that only ~15% of molecules of wild type APE1 and its mutants had a

catalytic activity towards this substrate. Probably, APE1 formed three types of complexes

with DHU-substrate, and only one of them was catalytically active. In NIR process the actions

of all enzyme forms were described by identical kinetic schemes, consisting of enzyme-

substrate complex formations, fast substrate cleavage, rapid isomerisation of the protein-

nucleic complex, slow conformational change resulting in the formation of stable enzyme-

product complex and product release from this stable complex. The process of product release

limited turnover of enzyme. The rate constant of DHU-substrate cleavage by APE1 in NIR

conditions was comparable with analogous values for tetrahydrofuran and AP containing

substrates in BER conditions (Timofeyeva et al. JBSD 26, 2009). The catalytic cleavage of

DHU-substrate by APE1ΔN61 and APE1K98A occurred ~500 and ~200 fold slower,

respectively, in comparison with APE1 demonstrating the critical role of REF1 domain and

Lys-98 in the catalysis in NIR.

The research was supported by grants from RFBR (08-04-12211, 10-04-00070),

SBRAS (28, 48), Russian Ministry of Education and Science (NS-3185.2010.4,

02.740.11.0079, 02.740.11.5012).

83

Ground- and Excited-State Recoordinations in Optical Molecular Sensors

Ushakov E.N.1, Dmitrieva S.N.2, Vedernikov A.I.2, Kuz’mina L.G.3, Gromov S.P.2

1 Institute of Problems of Chemical Physics, Russian Academy of Sciences, Chernogolovka, 2 Photochemistry Center, Russian Academy of Sciences, Moscow,

3 N.S. Kurnakov Institute of General and Inorganic Chemistry, Moscow

Interest in recoordination reactions of metal cations with the ligands having a donor-

acceptor chromophore arose in connection with the development of optical molecular sensors

and molecular photoswitches. Reversible noncovalent bond-breaking reactions in cation–

macrocycle complexes of ionochromic N-phenylazacrown compounds are most widely

investigated. In some works it is asserted that photoinduced disruption of the cation–nitrogen

coordination bond is followed by complete dissociation of the complex, i.e. the metal cation is

released from the macrocycle cavity into bulk solvent during the excited state lifetime of the

chromophore unit. However, this mechanism may be inapplicable for the systems with a

relatively short excited state lifetime.

Previously we supposed that the inclusion complexes of metal cations with

arylazacrown compounds can undergo ground-state recoordination, i.e. they can exist as two

thermodynamically equilibrated forms (like А and В in the scheme) that differ from each

other in the number of coordination bonds. However, until recently there were no solid

experimental evidences for this hypothesis.

In this report we will discuss the mechanisms of photoinduced and ground-state

recoordinations, as well as the kinetic aspects of cation–macrocycle interactions. In addition,

experimental data will be presented that conclusively prove the possibility for the ground-

state recoordination to occur in metal complexes of arylazacrown compounds.

This work was supported by the Russian Academy of Sciences and the Russian

Foundation for Basic Research.

84

Cucurbit[8]uril as Photocontrolled Molecular Assembler for [2+2]-

Autophotocycloaddition of Styryl Dyes in Aqueous Media

Vedernikov A.I.1, Sazonov S.K.1, Kuz’mina L.G.2, Churakov A.V.2,

Strelenko Yu.A.3, Howard J.A.K.4, Alfimov M.V.1, Gromov S.P.1

1 Photochemistry Centre of the RAS, Moscow, RF; 2 Institute of General and Inorganic Chemistry of the RAS, Moscow, RF;

3 Institute of Organic Chemistry of the RAS, Moscow, RF; 4 Chemistry Department, Durham University, Durham, UK

Styryl dyes of the 4-pyridine series 1 form pseudorotaxane-type complexes with

cucurbit[n]urils (CB[n], n = 7, 8) in water. The stoichiometry, structure, and stability of these

complexes were studied using optical and NMR spectroscopy, including NMR titration, and

X-ray diffraction. The dyes have strong absorption band at ~400 nm.

NOMe

OMeR

NOMe

OMeR

NMeO

MeOR

N

N

R

R

OMe

OMe

MeO

MeO

{1}2@CB[8]

+

1: R = Et, (CH2)3NH3+,

(CH2)3SO3-

+

+

h

+

+

- CB[8]

rctt-2

CB[8]

Cations of dyes 1 are prone to give syn-head-to-tail packing motif in solid state. Under

irradiation with visible light, thin polycrystalline films of the dyes undergo relatively slow

stereospecific [2+2]-photocycloaddition (PCA) reaction to form rctt-isomers of cyclobutanes

2. In water, dyes 1 and their complexes with CB[7] undergo only reversible E–Z

photoisomerisation. The efficient PCA reaction accomplishes within {1}2@CB[8]

termolecular complexes giving the only rctt-2. The stability of 1:1 complexes between CB[8]

and rctt-2 is lower than that of 1@CB[8]. This makes it possible to use CB[8] as a

supramolecular catalyst in order to attain complete photoconversion of 1. Thus, CB[8] is a

photocontrolled molecular assembler for cyclobutanes. Such systems can be used for optical

recording and storage of information, for creation of molecular machines driven by light.

This work was supported by the Russian Foundation for Basic Research, the Russian

Academy of Sciences, and the Royal Society of Chemistry (UK).

85

Porous homo- and heterochiral Co(II), Ni(II) malates and aspartates with

high thermal stability of metal–organic framework Yutkin M.P.1, Dybtsev D.N.1,2, Fedin V.P.1

1 Nikolaev Institute of Inofrganic Chemistry SB RAS, Russian Federation, 630090 Novosibirsk, Lavrentev av. 3, yutkin@niic.nsc.ru,

2 Division of the Advanced Materials Science, POSTECH, Pohang, Korea

Homochiral (enantiopure) porous metal-organic coordination polymers, possessing a

regular arrangement of optical and Lewis centers, are perspective as heterogeneous catalysts

and in separation of recemic mixtures onto individual enantiomers.

Following recently published synthetic method we characterize number of unique

families of isoreticular homochiral porous metal-organic coordination polymers with tunable

pore size and nature of metal centers. For example, a solvothermal reaction of Co2+/Ni2+ salts,

L-aspartic or L-malic acids with different N-donor bridging ligands such as 4,4'-dipyridil

(bpy) or trans-bis(4-pyridil)ethylene (bpe) afforded to the formation of homochiral porous

coordination polymers with general formula [M2(L*)2L]•xG (M = Co2+, Ni2+, L* = L-

aspartate/L-malate; L = bpy, bpe, G = different guests). All this compounds share the same

pillared-layer topology of metal-organic framework, thus the only general description of

structures will be made. The aspartate/malate anions acting as tridentate and bridging ligands

form chiral layers [M(L*)]. These layers are connected in two dimension via N-donor bridges

giving rise 3D metal-organic structures. Thus, the interlayer distance solely controlled by N-

donor liner length. As the consequence, the cross section of the channels and accessible

volume wary from 4 × 5 Å, 22% for [Co2(L-asp)2bpy] to 4 × 7 Å, 26% for [Co2(L-asp)2bpe].

In summary, the design abilities

of new synthetic scheme, which allow

to easily control pore size and nature of

metal centers in homochiral porous

metal-organic coordination polymers,

have been demonstrated. In addition

catalytic and sorption properties of

obtained compounds have been

investigated.

This work is supported by Russian Foundation for Basic Research (Grants 09-03-90414 and 09-03-12112).

86

Increase of effectiveness of fundamental research and innovations in high technology segment.

Popkov I. A.

President RUSCHEMBIO

Infrastructural project RUSCHEMBIO of Russian Corporation of Nanotechnologies

RUSNANO was established to solve the problem of prompt delivery of chemical and

biochemical reagents, consumables and high-tech equipment for research institutes and

commercial companies and advertising of their products to Russian and foreign market.

One of the priority tasks of RUSCHEMBIO is to create the established supplying of

reagents and consumables. The key units of this system will be the stock of domestic and

foreign products prompt delivery of products in whole Russia and dealing with reagents

requiring special temperature conditions and licenses. Understanding of customer needs and

coordination between supplies and manufactures is the key thing to create a stock with a good

range of goods. Received information from customers on reagents and consumables will help

to get important documents for custom clearance in advance.

We hope that join work will help to solve the problem of prompt delivery of reagents,

consumables and hi-tech equipment and the science will be on high level.

87

Roundtable

New possibilities of studies in functional proteomics and drug design: applying matrix SPR technology

Bio-Rad Laboratories

Roundtabe dedicated to discussion of new possibilities for high-throughput proteomics

experiments and its impact on research progress in functional proteomics and drug design.

Topics to be discussed: Protein-protein interaction and interfaces, receptor-ligand kinetics,

approaches to study expression regulation by DNA-bindning proteins.

The rapid acquisition of accurate protein interaction data is a vital need in the

investigation of many biological systems. The rapidly expanding field of proteomics, for

example, demands reproducible, robust, high-performance methods to supplement traditional

technology in the interrogation of the immense network of protein interactions in a cell.

New approaches to high-throughput SPR experiments using matrix SPR technology will

be presented: methodological approaches of protein-protein interface mapping will be

presented with experiments on reconstruction of BLIP–TEM1 beta-lactamase interface;

studies of IFN receptor/IFN-alpha interactions showed how these studies can lead to highly-

effective anticancer drug development; experiments on DNA-protein and RNA-protein

interactions demonstrate new insights to expression regulation in a cell.

Impact of new matrix SPR technology of ProteOn XPR36 system on research

effectiveness and productivity will be discussed.

Author index

91

A Adgibeck D. 16

Aleksandrova N.A. 73 Alekseeva I.V. 56

Alfimov M.V. 25, 42, 62, 73, 84 Ambrosek D. 14 Ame J.-C. 70 Antipin I. 21 Arnaud-Neu F. 37

Assfeld X. 14 Atabekyan L.S. 46, 55 B Bagatur’yants A.A. 59, 69 Bagryanskaya E. 9, 67, 74 Baouz S. 57 Basok S.S. 59 Batat P. 29 Beckert B. 45 Beniaminov A. 39 Berdonosova E.A. 77

Billard I. 10, 65 Boutorine A. S. 11 Bulygin K. 24, 33, 57 Burdin A.A. 44 C Cadot E, 12, 35, 61 Chernolovskaya E.L. 13, 40 Chernonosov A.A. 58 Chibisov A.K. 46, 55 Churakov A.V. 84 Churakova M.V. 59 Comte C. 17 D Daniel C. 14 Demisheva I.V. 42 Dianov G. 15 Dmitrieva S.N. 59, 69, 83 Doluca O. 11 Dontsova O. 16 Duval S. 61 Dyakonova E.S. 31 Dybtsev D.N. 77, 85

E Efremov A.60, 80 Entelis N. 17

92

F Fedin V. 9, 18, 38, 51, 66, 67, 74, 78, 85 Fedorenko E.V. 68 Fedorova O. S. 19, 31, 56, 58, 71, 72, 82 Ferlay S. 21 Filichev V. V. 11 Floquet S. 12, 35, 61 Fomina M. V. 62 Freidzon A.Ya. 59, 69 Frolova L. 33 G Gabibov A.G. 56 Gaillard C. 65 Giegé R. 22 Gehin A. 21 Georg S. 65 Gerasko O. 9, 38, 74 Golovin A.V. 48 Gorbatchuk V.V. 23 Grachev E.V. 44 Graifer D. 24, 32, 33 Grigor'ev I. 74 Gromov S.P. 25, 55, 59, 62, 69, 73, 83, 84 Gruner M. 47 Gubaidullin A.T. 47 H Habicher W. 47 Haouas M. 35, 61 Heckel A.-M. 17 Hedegaard M.M. 45 Hijazi A. 61 Hosseini M.W. 21, 26 Hountondji C. 57 Howard J.A.K. 84 Huc I. 27 Hyunuk Kim 77 I Ildyakov A.V. 44 Ilina E. S. 28, 34 Ilyin A.A. 63 Ishchenko A. A. 82 Iskhakova I.T. 73 Ivanov A. V. 64 Ivanov D.A. 46 J Jonusauskas G. 29

93

K Kalchenko V. 10, 30, 37, 68 Kaliya O. 71 Kanazhevskaya L.Yu. 31 Karpova G. 24, 32, 33, 36, 43, 57, 63, 64 Karpova T. 75, 81 Katsyuba S.A. 47 Keita B. 61 Khramtsov V. 9 Khairulina Y. 24, 33 Khodyreva S. N. 28, 34, 70 Kholdeeva O.A. 65 Kimoon Kim 77 Kirilyuk I. 9, 74 Kleshnina S.R. 47 Klimchuk O. 65 Knorre D. 71 Komarov V.Yu. 44, 75, 76 Konovalov A. 47 Kopylov A.M. 48 Korda T. 37 Korenev V. 12, 35 Korotaev E.V. 68 Kossinova O.A. 36, 43 Kostin G. 37, 68 Koval O.A. 79 Koval V.V. 31, 56, 72, 82 Kovalenko E. 38 Kovalenko K.A. 66 Kozlova M. 21 Krasikova Y.S. 41 Krumkacheva O. 67 Krol A. 36, 39 Kruglova N. S. 40 Kryuchkova N.A. 68 Kuchnov D.S. 13 Kuligina E.V. 79 Kuratieva N.V. 76 Kurchavov N.A. 59, 69 Kutaev N.V. 44 Kutuzov M.M. 34, 70 Kuzmina L.G. 25, 62, 69, 83, 84 Kuznetsov N. 72 Kuznetsova A. 71 L Lavie-Cambot A. 29 Lavrik O. I. 28, 34, 41, 70 Le Caer J.-P. 57

94

Leclerc N. 12 Livshits V.A. 42 Lobova N.A. 55, 73 Logvina N. 16 Loktev V.B. 43 Loos P. –F. 14 Luk'yanets E.A. 58, 71 Lutay A. 52 M Maksimchuk N.V. 66 Malygin A.A. 36, 43, 63, 64 Manakov A.Yu. 44, 75, 81 Marque S. 67 Marrot J. 35 Martin R. P. 17 Masquida B. 45 Mazalov L.N. 68 Mbomekallé I.-M. 35 McClenaghan N.D. 29 Meschaninova M. 17, 40 Miroshnichenko S. 10 Mustafina A. 47 N Nadjo L. 61

Nechaev S. 52 Nielsen H. 45 Nikiforov A. S. 62 Ngo Biboum R. 61 O Ogienko A.G. 44 Ouadi A. 10, 65 Ovsyannikov A. 21 P Petrov N.Kh. 46 Plyusnin V.F. 67 Polovyanenko D. 9, 67, 74 Popkov I. A. 86 Pyshny D.V. 13, 17, 60 R Rassokhin T.I. 48 Rechkunova N.I. 41 Richter V.A. 79 Rodionova T.V. 44, 75, 76 Rogoza A. 60

95

Röder B. 58 Rubtsova M. 16 S Samsonenko D.G. 77, 78 Saparbaev M. K. 82 Sapchenko S.A. 78 Sazonov S.K. 84 Scherbakova D. 16 Schreiber V. 70 Shatsky I.N. 43 Shubernetskaya O. 16 Semenov D.V. 79 Semenov S. 9, 74 Serikov R. 60, 80 Sidorenko N.I. 69 Simonnet-Jegat C. 12, 61 Skiba S. S. 81 Skripacheva V. 47 Skvotsov D. 16 Smekalova E. 16, Smirnov A. 17 Soldatov D.V. 81 Solodovnikov S.F. 76 Solov'eva L.I. 58, 71 Solovieva S.E. 21, 47 Spiridonova V.A. 48 Stenin Y.G. 81 Stepanov G. A. 79 Stoporev A.S. 44 Strelenko Yu.A. 84 Sukhanova M.V. 34 Surdina A.V. 48 T Tarassov I. 17 Taulelle F. 35, 61 Terekhova I.S. 44, 81 Timofeyeva N. A. 82

Torgov V. 37, 68 U Us T. 37 Ushakov E.N. 25, 83 V Varnek A. 49 Vedernikov A.I. 25, 55, 59, 62, 69, 73, 83, 84 Venyaminova A. 17, 24, 32, 33, 40 Villevald G. 75, 81 Vives G. 29

96

Vlassov V.V. 13, 40, 50, 52, 80 Voronina L.V. 42 Vostrikova K. 51 W Westhof E. 39 Woisard A. 57 Y Yanshina D.D. 63 Yutkin M.P. 85 Z Zenkova M.A. 13, 40, 52, 60, 80 Ziganshin M.A. 23 Zvereva E.E. 47 Zvereva M. 16

ВСЕ ДЛЯ ВАШЕЙ ЛАБОРАТОРИИ

Компания «Промикс» создана в 1993 году для обеспечения научно-

исследовательских лабораторий РАН, РАМН и Россельхозакадемии и клинических лабораторий лечебных учреждений оборудованием и расходными материалами для надежных, высококачественных и точных лабораторных исследований. На протяжении 17-ти лет деятельность компании направлена на внедрение современных методов исследований в практику российских лабораторий. С самого начала нашей деятельности мы сделали сознательный выбор в пользу качества поставляемого нами оборудования, поэтому мы уже много лет сотрудничичаем с производителями, чья продукция высоко котируется у специалистов во всем мире. Основные принципы нашей работы — профессионализм и качество услуг. В настоящее время по объему предоставленных услуг на лабораторном рынке ООО «Промикс» является лидером среди компаний Сибирского региона.

НАША ПРОДУКЦИЯ На сегодняшний день ООО «Промикс» является поставщиком более 160 российских, европейских, азиатских и американских заводов-производителей, а ассортимент предлагаемой продукции превышает 6000 наименований. Для многих из них ООО «Промикс» является авторизованным дилером на территории Сибири и Дальнего Востока

Основные партнеры для научного направления (производители):

1. Thermo Fisher Scientific (США) — спектральное оборудование,

системы для выделения ДНК/РНК/белков, гистологическое оборудование Microm и Shendon, дозаторы, пластик (Thermo, Nalgene и Nunc) и др. достойное оборудование, которое сейчас предлагает эта крупнейшая мировая компания

2. Beckman Coulter (США) — центрифуги, системы проточной цитометрии, спектрофотометры, капиллярный электрофорез, анализаторы клеток и частиц, секвенаторы и др. высокотехнологичное оборудование

3. General Electric Healthcare (США) — все для хроматографии производства Pharmacia (Швеция), фильтрующие системы производства Whatmann, спектрофотометры GE

4. Bio-Rad (США) — оборудование для электрофореза, хроматографии, ПЦР

5. Millipore (США) — системы очистки воды для любых типов лабораторий и потребностей

6. BMT (Чехия) — термостаты, стерилизаторы, автоклавы 7. BioSan (Латвия) - персональное лабораторное оборудование для

пробоподготовки в области генной инженерии и биотехнологии

E-mail: promix@promix.ru www.promix.ru

ООО «Промикс» 630117, Новосибирск, а/я 197 ул. Арбузова, 6, офис: 4, 5, 6 т/ф: (383) 332-80-26, 336-01-66 www.promix.ru

8. Карл Цейсс (Германия) — микроскопы от учебных до исследовательских моделей (световая, электронная, конфокальная микроскопия)

9. Foss Electric (Дания)- анализаторы для контроля качества мясной, молочной продукции, комбикормов, продуктов зернопереработки и сырья

10.ЛоиП (Россия) — лабораторная мебель европейского качества, обеспечивает безопасность работы со всем спектром реагентов, которые могут встретиться в практике и оборудование для анализа нефтепродуктов.

ООО «Промикс» как дилер осуществляет:

консультационную помощь при выборе оборудования продажу оборудования поставку расходных материалов и реагентов техническую и методическую поддержку

НАШИ КЛИЕНТЫ Наши основные поставки осуществляются на территории Западной и Восточной Сибири + Тюменская область (включая ХМАО). Нашими клиентами являются:

НИИ Сибирских отделений РАН, РАМН и Россельхозакадемии учреждения Роспотребнадзора пищевая промышленность, фармпроизводство лечебно-профилактические учреждения МЗ РФ и ФМБА частные клиники и диагностические центры

ИНФРАСТРУКТУРА ООО «ПРОМИКС» В настоящий момент в компании работают более 40 сотрудников преимущественно с высшим химическим, биологическим, медицинским и техническим образованием. Компания обладает собственными складскими помещениями, транспортной службой, таможенным отделом. В структуре фирмы важное место занимает служба сервисного обслуживания, что позволяет обеспечивать запуск в эксплуатацию лабораторной техники, обучение персонала, гарантийное и постгарантийное обслуживание. Длительный положительный опыт работы, высокая профессиональная квалификация сотрудников, широта ассортимента, оптимальное соотношение цена-качество предлагаемой продукции, информационная и техническая базы помогают компании сохранять надежные и стабильные отношения с клиентами и удерживать лидерские позиции на региональном рынке лабораторного оборудования.

Notes

Коллектив авторов, 2010

Сборник трудов международной конференции

Supramolecular Chemistry for Materials and Life Sciences

June 29 – July 3, 2010

Novosibirsk, Russia

Рецензенты академик РАН Власов В.В.

к.б.н. Рихтер В.А.

Подготовка оригинал-макета, редактирование Малыгин А.А.

Подписано в печать 07.06.2010 Формат 70x100/16. Бумага офсетная. Усл. Печ. Л. 6.25

Заказ № 158. Тираж 100 экз.

Отпечатано в типографии ЗАО ИПП «Офсет» 630117 Новосибирск, ул. Арбузова, 4а