Synthesis of phosphitylated compounds using quaternary heterocyclic activator

FIELD: chemistry.

SUBSTANCE: method involves reaction of a hydroxyl-containing compound, which contains a sugar fragment, with a phosphitylation agent in the presence of an activator of formula I , where R = alkyl, cycloalkyl, aryl, aralkyl, heteroalkyl, heteroaryl, R1, R2 = either H, or together form a 5-6-member ring, X1, X2 = independently N or CH, Y = H or Si(R4)3, where R4 = alkyl, cycloalkyl, aryl, aralkyl, heteroalkyl, heteroaryl, B = trifluoroacetate, dichloroacetate, mesyl, tosyl, o-chlorophenolate.

EFFECT: novel method of producing phosphoramidites.

17 cl, 21 ex

 

The technical field to which the invention relates

The present invention relates to methods for hospitalizovanih compounds using specific activators, particularly, to the synthesis of phosphoramidites.

Prior art

Oligonucleotides are key compounds in the life Sciences that have important values in various fields. Them, for example, are used as probes in the analysis of gene expression, as primers in PCR or DNA sequencing.

In addition, there are also a number of potential therapeutic applications, including, for example, the antisense oligonucleotides.

A number of chemical modifications introduced into the oligonucleotides to increase their applicability in diagnosis, as research reagents and as therapeutic agents, for example, to stabilize against nucleases.

The synthesis of oligonucleotides can be performed using both methods in the solution phase and solid-phase methods. The preferred method at present is a method by solid-phase synthesis, in which the oligonucleotide get on a solid substrate and oligonucleotide increasing by successive addition of nucleotides.

A growing number of applications require higher is richest oligonucleotides; therefore, there is an urgent need to develop improved synthetic method.

For an overview, see e.g., “Antisense - From Technology to Therapy” Blackwell Science (Oxford, 1997).

One known type of building blocks in the synthesis of oligonucleotides is phosphoramidite; see, for example, S.L. Beaucage, M.H. Caruthers, Tetrahedron Letters 1859 (1981) 22. These phosphoramidite nucleosides, deoxyribonucleosides and derivatives of these and others are available for purchase. In conventional solid-phase synthesis is used 3'-O-phosphoramidite, but other synthetic methods are also used 5'-O and 2'-O-phosphoramidite. One stage in obtaining these phosphoramidite nucleosides is fosfaurilirovania (protected) nucleosides. Most often, hydroxyl group and amino group and other functional groups present in the nucleoside, protect before fosfaurilirovaniem the remaining 3'-, 5'- or 2'-O hydroxyl group. There are several ways to obtain Monomeric (nucleosides) and polymer (nucleotides or oligonucleotides) of phosphoramidite. Known methods lead very often to problems in chemistry or security. For the application of this chemistry for large-scale synthesis of (100 kg - 1000 kg) cost efficiency the need to improve.

Traditionally, fosfaurilirovania nucleosides carry out the processing of protected NUS is esedov hospitaleros reagent, such as chloro-(2-cyanoethoxy)-N,N-diisopropylaminoethyl, which is very reactive and does not require an activator or 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (bis FOS or bis-humidity reagent), which requires activator.

Promoters most commonly used in reactions of fosfaurilirovania is 1N-tetrazol.

For 1N-tetrazole there are inherent problems, especially when conducting large-scale synthesis. For example, we know that 1N-tetrazol is explosive and toxic. In accordance with the safety instructions (MSDS) 1N-tetrazol (1N-tetrazol, 98%) may have harmful effects through inhalation, absorption inside and absorption through the skin.

In addition, 1N-tetrazol is expensive, especially when large-scale synthesis it has a significant impact on costs in the synthesis of oligonucleotides.

The MSDS also States that 1N-tetrazol may explode if heated above its melting point equal to 155°C, and can form a very sensitive explosive compounds with metals. In the case of large-scale synthesis reactor 1N-tetrazol will represent a great danger to people and the environment.

In addition, it is known that 1N-tetrazol requires special treatment the deposits during storage, application and utilization.

1N-tetrazol and related derivatives, such as 5-ethylthio-1N-tetrazole, 5-benzylthio-1N-tetrazol also have the ability to destroy a target molecule. Therefore, the cleavage is sensitive to acid protective group described in various publications (Krotz et al, Tetrahedron Letters, 1997, 38, 3875).

Adverse removing cyclotouring protective group is also known with the use of chloro-(2-cyanoethoxy)-N,N-diisopropylaminoethyl. In addition to the tendency of detachment of the applicable protective groups this hospitalise agent will lead to large amounts of 3'-3'-isomers. The resulting humidity must be purified by chromatographic stage with high time and material costs.

Especially in application to fosfaurilirovania of oligomeric phosphoramidites known methods result in the result, mainly, to the decomposition or complex mixtures of target molecules and by-products.

The use of bis-FOS with some activators are generally known for Monomeric nucleoside of amidites, but in the case of oligonucleotides low reactivity makes this approach very complicated.

Low reactivity leads to long reaction time (2-6 h). Attempt to avoid long reaction time will require a broad range the aqueous excess hospitilised agent and activator. At the end of this management response will also require additional stages of refinement.

EP 0906917 A2 and Hayakawa et al., J. Am. Chem Soc. 120 (1998) 12395-12401 disclose the use of the triflate imidazole for the synthesis of phosphoramidites. The yield and purity of the described synthesis could not be repeated.

Furthermore, the method Hayakawa was carried out with the use of the activator, which was obtained, isolated and purified separately. After cleaning sensitive to water activator is necessary to store the activator in absolutely dry conditions.

Sensitivity and low reactivity of the activator will result in complicated manipulation, which is difficult for large-scale synthesis of amidites.

In all experiments with this activator Hayakawa resulting humidity should be cleaned by high-cost chromatographic stage.

However, in all cases, the reaction of fosfaurilirovania was incomplete and ineffective, and therefore, the cleaning stage is always a basic requirement.

Fosfaurilirovania sensitive oligonucleotides ends, mainly decay.

The outputs and the degree of purity of the described synthesis can not be repeated, due to the fact that the applied triflate imidazole have vysokooleofobnyh nature and high gigroskopicheskoi ten is enciu. These properties in the final result when working with the basic quantities lead to decomposition and hydrolysis. Described activators isolated and used in their pure form.

This method of synthesis of amidites requires flash chromatography for purification of the target compounds.

In addition, Hayakawa used this connection to education mezhnukleotidnyh communication (condensation of amidite with nucleoside).

Hayakawa et al., J. Org. Chem. 61 (1996) 7996-7997 discloses the use of the triflate benzimidazole for condensation of phosphoramidite with nuke.

Hayakawa et al., J. Am. Chem. Soc. 123 (2001) 8165-8176 discloses the use of complexes of acid/azole for condensation of phosphoramidite with nuke.

Arnold et al., Collect. Czech. Chem. Commun. 54 (1989) 523-532 discloses an automated chlorinity and humidity synthesis of oligodeoxyribonucleotides and inter alia the use of 1-methylimidazole in condensation of phosphoramidite with nuke.

Summary of the invention

The aim of the present invention is to provide a method of obtaining hospitalizovanih compounds which overcome at least some of the disadvantages of the prior art.

An additional aim of the invention is to provide an activator having improved properties compared with the activators of the prior art.

An additional aim of izaberete the Oia is to provide a mixture of the activator/additive, having improved properties compared with the activators of the prior art. In one aspect the present invention provides a method of obtaining fosfaurilirovannogo connection, comprising the stage of:

interaction of hydroxyl-containing compounds with hospitaleros agent in the presence of an activator having the formula I

where

R = alkyl, cycloalkyl, aryl, aralkyl, heteroalkyl, heteroaryl

R1, R2= or H, or together form a 5-6-membered cycle,

X1, X2= independently or N, or SN,

Y = H or Si(R4)3where R4= alkyl, cycloalkyl, aryl, aralkyl, heteroalkyl, heteroaryl,

B = deprotonirovannoi acid.

The activator can be used stoichiometrically or catalytically (3-50 mol.%, preferably 10-30 mol.%) or in excess (up to 300 mol.%).

In a preferred embodiment, the activator has the formula selected from the group consisting of

where

Y defined above,

R represents methyl, phenyl or benzyl.

These activators are, for example, described in Hayakawa et al, J. Am. Chem. Soc. 123 (2001) 8165-8176.

In one embodiment, the activator is used in combination with the additive. The additive can be selected from deprotonirovannoi form compounds having formula I, and other heterocycle the definition of grounds for example pyridine. The appropriate relationship between the activator and the additive is from 1:1 to 1:10.

In one preferred embodiment, the activator can be obtained by following the method of “in situ”. In this case, the activator is selected, which leads to improved results. Hydrolysis or decomposition of the target molecules is suppressed.

For fosfaurilirovania high yield 3'- and/or 5'-position of the oligonucleotides (di-, tri-, Tetra-, Penta-, hexa-, hepta - and oktamery) obtaining in-situ activator and mix with the additive is preferred.

As described above, fosfaurilirovania is especially applicable in the synthesis of oligonucleotides and the building blocks of phosphoramidites. Therefore, in a preferred embodiment, the hydroxyl-containing compound contains a fragment of sugar, such as a nucleoside or an oligomer, which is its derivative. Such nucleosides are, for example, adenosine, cytosine, guanosine and uracil, deoxyadenosine, deoxyguanosine, deoxythymidine, detoxication and their derivatives, optionally containing protective groups.

The method of the present invention is particularly applicable to fosfaurilirovania oligonucleotides (di-, tri-, Tetra-, Penta-, hexa-, hepta - and oktamery). Such fosfaurilirovania oligonucleotides are used, for example, in the synthesis of KRU is different oligonucleotides through the concept of fragment condensation.

Usually, they appropriately protect their heterocyclic functionality and hydroxylases groups, with the exception of one, you want to hospitalizati. In a typical case, dimethoxytrityl, monomethacrylates or siliconera protective group (e.g., TBDMS) are used as protective groups for 5 ON group, providing fosfaurilirovania 3'-Oh group.

Also, 3'-Oh group can be protected by a protective group (LEV, TBDMS, etc.) and unprotected 5'-HE will spend the 5'-O-fosfaurilirovania nucleosides or nucleotides.

Methods fosfaurilirovania can be used in the synthesis of 3'- or 5'-phosphoramidites with identical results.

The resulting target molecule reactions fosfaurilirovania represents in one embodiment, phosphoramidite and has the structure:

Z represents a leaving group, for example, CH3C2H5CH2C6H5, -CH2CH2CN, -CH2CH=CHCH2CN, para-CH2C6H4CH2CN, -(CH2)2-5N(H)COCF3, -CH2CH2Si(C6H5)2CH3or-CH2CH2N(CH3)COCF3and where R3represents alkyl having from 1 to about 6 carbon atoms; or R3represents heterologously or heterocycle is alkanniny cycle, containing from 4 to 7 atoms and having up to 3 heteroatoms selected from nitrogen, sulfur and oxygen, and “connection” represents the rest of hydroxyacetamido connection, for example a nucleoside, nucleotide or oligonucleotide.

In this case, the atom P(III) bonded to two oxygen atoms (or two forms of communication R-Oh) and one nitrogen atom (forming one relationship between P-N)which belongs to the amino group, preferably, Diisopropylamine, diethylamine or other secondary amines.

The reaction of condensation of phosphoramidite with another hydroxyl group of the other molecule (compound A) will lead to a complex trifiro of phosphite patterns:

In this case, the atom P(III) has connections with three oxygen atoms (forming three bonds R-Oh) and has no connection with nitrogen.

In General, hospitalise agent may be the same as in the reactions of fosfaurilirovania using 1 - N-tetrazole.

In the preferred embodiment, it has the formula

where Z represents a leaving group, for example, CH3C2H5CH2C6H5,

-CH2CH2CN, -CH2CH=CHCH2CN, para-CH2C6H4CH2CN, -(CH2)2-5N(H)COCF3,

-CH2CH2Si(C6H5)2CH3or-CH2 CH2N(CH3)COCF3and R1and R2independently represent a secondary amino group, N(R3)2where R3represents alkyl having from 1 to about 6 carbon atoms; or R3represents heterologously or heterocyclicamines cycle containing from 4 to 7 atoms and having up to 3 heteroatoms selected from nitrogen, sulfur and oxygen.

Typical hospitalise agent is a 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite.

Other preferred hospitalinaya agents are derivatives of oxaphosphorin, as described in N. Ok et al., J. Am. Chem. Soc. 2003, 125, 8307-8317 included as references. This hospitalise agent allows the synthesis of oligonucleotides, where mezhnukleotidnyh connection can be converted to phosphothioate stereoselective manner. Such diastereoselective synthesized mezhnukleotidnyh phosphothioate relations have a promising value for the application of phosphothioate as antisense drugs.

Suitable examples of deprotonated acid-are triplet, triptorelin, dichloracetate, mesyl, tosyl, o-chlorophenolate. Acids with a pKa of less than 4.5 are preferred. Preferably, they are of low nucleophilicity.

In one embodiment, the reaction is carried out in PR is the absence of molecular sieves or other water-bonding agents. In General, water should be deleted or record through the drying medium during the reaction.

It is possible to combine the activator of the present invention with hospitaleros agent and add hydroxyl component later. It is also possible to combine the activator with hydroxyl-containing compound and then add hospitalise agent.

In the case of use of the additive, the activator is mixed with hydroxynonenal before adding hospitilised agent.

To generate activator “in situ” of the selected acid is preferably added after the addition of the additive under conditions of controlled temperature reactions.

Hospitalise agent may be added before adding the selected acid or after you have added.

In relation to adding acid and hospitalised agent nucleoside component can be added at the end or in the beginning.

In a preferred embodiment, the corresponding base activator, hydroxyl-containing compound and hospitalise agent are combined and the acid is added to start the reaction.

An additional object of the invention is to use an activator having the formula I

where

R = alkyl, cycloalkyl, aryl, aralkyl, heteroalkyl, heteroaryl,

R1, R2= or H, or together form a 5-6-membered cycle,

sub> 1, X2= independently or N, or SN,

Y = H or Si(R4)3where R4= alkyl, cycloalkyl, aryl, aralkyl, heteroalkyl, heteroaryl,

B = deprotonirovannoi acid,

as activator for fosfaurilirovania hydroxyl-containing compounds hospitaleros agent.

An additional aim of the invention is the combination of activator and neprolongirovannogo Foundation (supplements)that will create a balance between the two types. The resulting equilibrium exhibits superior properties in comparison with the activators of the prior art.

Especially in connection with the use of acetone activator/catalyst would not be known adverse reactions (decomposition or the formation of 3'-3'or 5'-5'-homologs). Acetone also has the ability to dissolve educti and reagents.

In accordance with the prior art, in the case of long reaction time release Diisopropylamine and the presence of activated Bis-FOS leads to the decomposition of the target compound (datetimerange, CE-splitting, deponirovanie or removal of other protective groups). The presence of acetone and specific activator reduces data trends.

The presence of acetone dampens the activity of any number of Diisopropylamine (DIPA), which is released in p is acesse fosfaurilirovania. It can be used to fosfaurilirovania shorter or longer oligonucleotides with similar results (without decomposition). Other ketone compounds having the formula Rx-C(=O)-Rywhere Rxand Ryindependently represents a C1-C6alkyl or together form cycloalkyl, can also be used because they are capable to form enolate in the presence of, for example, amines, having CH2group in α-position.

In addition, the use of acetone leads to a longer reaction time without removal of 5'-O-protective group. In both cases, the use of acetone will protect various protective groups and avoid trends deponirovanie.

Acetone also has a better toxicity profile and improved environmental properties compared with, for example, acetonitrile and is inexpensive.

An additional goal, therefore, is to use acetone as a reaction medium or co-solvent in the synthesis of phosphoramidites.

The combination of the activator with a certain amount of additives supports more efficient way of fosfaurilirovania longer and sensitive oligonucleotides (3' or 5' without protective groups).

Typically, the reactivity of the reagent is increased, so that the synthesis was over in about 2-5 is in.

When using the methods of the present invention eliminates the need for additional purification stages.

The resulting Monomeric and oligomeric humidity can be used for solid - and liquid-phase synthesis of oligonucleotides.

Activator, or a combination of activator/additive are particularly applicable for the synthesis of phosphoramidite adenosine, phosphoramidite cytosine, phosphoramidite guanosine and phosphoramidite uracil, phosphoramidite deoxyadenosine, phosphoramidite of deoxyguanosine, phosphoramidite of deoxythymidine, phosphoramidite deoxycytidine and phosphoramidate oligonucleotides having the formula Xnwhere each X is selected from A, dA, C, dC, G, dG, U, dT and n=2 to 30, preferably 2 to 12, more preferably 2 to 8 or 2 to 6 and their derivatives containing protective groups.

As used herein, the term oligonucleotide also includes oligonucleotide, analogs of oligonucleotides, modified oligonucleotides, nucleotide mimetics, etc. in the form of RNA and DNA. In General, these compounds contain a backbone of linked Monomeric subunits, where each linked Monomeric subunit directly or indirectly attached to the fragment of the heterocyclic bases. Linkages between Monomeric subunits, Monomeric subunit and fragments of heterocyclic bases mo is ut to change the structure, allowing the formation of many options for the resulting connection.

Modifications known in the field, represent a modification of the heterocyclic bases, sugars or links connecting the Monomeric subunit. Variations mezhnukleotidnyh ties, for example, described in WO 2004/011474 since the end of page 11, and is included as a reference.

Typical derivatives are phosphorotioate, phosphorodithioate, methyl and alkylphosphonate and phosphonoacetate.

Additional typical modifications have occurred fragment sugar. Or ribose as opposed to various sugar or one or more of the provisions supersede other groups such as F, O-alkyl, S-alkyl, N-alkyl. Preferred options for implementation are 2'-methyl and 2'-methoxyethoxy. All these modifications are known in this field.

Regarding the fragment of the heterocyclic bases, there are a number of other synthetic substrates that are used in this field, for example, 5-methylcytosine, 5-hydroxymethylcytosine, xanthine, gipoksantin, 2-aminoadenine, 6 - or 2-alkyl derivatives of adenine and guanine, 2-thiouracil. Such modifications are also disclosed in WO 2004/011474 starting on page 21.

When used in the synthesis of these grounds usually have a protective group, for example, N-6-benzyladenine, N-4-benzylation or N-2-isobutyl is guanine. In General, all reactive groups whose participation in further response is not provided, must be protected, especially hydroxyl groups of sugar.

In the variants of implementation related to the synthesis of oligonucleotide phosphoramidite is useful to carry out the reaction in the presence of acetone or other ketones, such as acetone, butanone, pentanone, hexanone, cyclohexanone, which can be used either as a reaction medium or as a co-solvent for the other solvents.

The invention is further illustrated by the following non-limiting examples.

Example 1

Synthesis of 5'-O-DMTr-T-3'-O-phosphoramidite using methylimidazole of triptoreline

5.0 g of 5'-O-DMTr-T-3'-Oh (9.2 mmol, 1.0 EQ.) and of 2.34 g of methylimidazole of triptoreline (11,9 mmol, 1.3 EQ.) dissolved in 100 ml of dichloromethane and add 3 g of molecular sieves 3E and the mixture is stirred for 10 minutes Added to 3.8 ml of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (11.9 ml, 1.3 EQ.). The reaction takes place completely in 2 hours Yield (determined by HPLC): 95%.

Example 2

Synthesis of 5'-O-DMTr-dGiBu-3'-O-phosphoramidite using benzylimidazole of triptoreline

322 mg of metronidazole of triptoreline (1,64 mmol, of 1.05 equiv.) and 1.0 g of 5'-O-DMTr-dGiBu-3'-HE (1.56 mmol, 1.0 EQ.) dissolved in 10 ml of dichloromethane and dobavlaut 500 mg of molecular sieves 3E. After 30 min of 0.52 ml of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (1,64 mmol, of 1.05 equiv.) and 0.1 ml of acetone added to the mixed solution. The reaction takes place completely in 30 minutes Yield (determined by HPLC): 74%.

Example 3

Synthesis of 5'-O-DMTr-dCBz-3'-O-phosphoramidite using methylimidazole of triptoreline

of 9.51 g of 5'-O-DMTr-dCBz-3'-HE (15 mmol, 1.0 EQ.) dissolved in 80 ml of acetone and 80 ml of acetonitrile. of 6.17 g of methylimidazole of triptoreline (32 mmol, 2.1 EQ.) and for 9.64 g of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (32 mmol, 2.1 EQ.) add to the mix the solution. The reaction takes place completely after 30 minutes Add 500 ml of ethyl acetate, the solution is extracted with twice 250 ml of NaHCO3solution and 250 ml of saturated salt solution. The organic layer is dried with MgSO4and evaporated to dryness. The residue is dissolved in 40 ml of dichloromethane, add 250 ml of pentane, the supernatant decanted and the residue is dried under reduced pressure with the formation of a colorless foam. Output (12.0 g, 14.4 mmol): 96%, purity (determined by HPLC): 93%.

Example 4

Synthesis of 5'-O-DMTr-dABz-3'-O-phosphoramidite using benzylimidazole of triptoreline

38 mg of benzylimidazole of triptoreline (0.14 mmol, 1.5 EQ.) dissolve in 5 ml of acetonitrile and add 300 mg of molecular sieves 3E. Add 145 μl of 2-cyanoethyl-N,N,N',N'-tetrazepam is peltoperlidae (0.46 mmol, 5.0 EQ.). After 30 minutes add 61 mg of 5'-O-DMTr-dABz-3'-OH (0.09 mmol, 1.0 EQ.) and the solution is stirred over night. The reaction takes place completely within 17 hours. Output (determined by HPLC): 91%.

Example 5

Synthesis of 5'-O-DMTr-dCBz-3'-O-phosphoramidite using catalytic amounts of methylimidazole of triptoreline

500 mg of 5'-O-DMTr-dCBz-3'-OH (0,79 mmol, 1.0 EQ.) dissolved in 18 ml of dichloromethane and 1 ml of DMF, add 3 g of molecular sieves 3E. 50 mg of metronidazole of triptoreline (0,17 mmol, 0.2 EQ.) and 276 μl of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (0.87 mmol, 1.1 EQ.) add to the mix the solution. The reaction takes place completely in 24 hours the Output (determined by HPLC): 89%.

Example 6

Synthesis of 5'-O-DMTr-dGiBu-3'-O-phosphoramidite using catalytic amounts of benzylimidazole of triptoreline

5 mg of benzylimidazole of triptoreline (0.02 mmol, 0.2 EQ.) dissolve in 5 ml of acetonitrile and add 300 mg of molecular sieves 3E. 145 μl of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (0.46 mmol, 5.0 equiv.) add to the mix the solution. After 1 h, add 60 mg of 5'-O-DMTr-dGiBu-3'-OH (0.09 mmol, 1.0 EQ.) and the solution is stirred over night. The reaction takes place completely within 48 hours. Output (determined by HPLC): 90%.

Example 7

Synthesis of 5'-O-DMTr-T-3'-O-phosphoramidite used the eat catalytic amounts of benzylimidazole of triptoreline

50 mg of benzylimidazole of triptoreline (0.18 mmol, of 0.18 EQ.) and 500 mg of 5'-O-DMTr-T-3'-OH (0,92 mmol, 1.0 EQ.) was dissolved in 28 ml of dichloromethane and add 3 g of molecular sieves 3E. 350 ál of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (1.0 mmol, 1.1 EQ.) add to the mix the solution. The reaction takes place entirely through 25 hours. Output (determined by HPLC): 90%.

Example 8

Synthesis of 5'-O-DMTr-T-R(S)-dCBz-3-phosphoramidite using methylimidazole of triptoreline

100 mg of 5'-O-DMTr-T-R(S)-dCBz-3'-OH (0.10 mmol, 1.0 EQ.) and 24.4 mg of metronidazole of triptoreline (0.11 mmol, 1.1 equiv.) dissolved in 10 ml of dichloromethane, add 200 mg of molecular sieves 4E. 32 μl of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (0.10 mmol, 1.0 EQ.) add to the mix the solution. The reaction takes place completely within 24 hours. Output (determined by HPLC): 60%.

Example 9

Synthesis of 5'-O-DMTr-dCBz-P(S)-dGiBu-3'-O-phosphoramidite using methylimidazole of triptoreline

100 mg of 5'-O-DMTr-dCBz-P(S)-dGiBu-3'-HE (0.09 mmol, 1.0 EQ.) and 17.8 mg of metronidazole of triptoreline (0.09 mmol, 1.0 EQ.) dissolved in 10 ml of dichloromethane, add 200 mg of molecular sieves 4E. 28 μl of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (0.09 mmol, 1.0 EQ.) add to the mix the solution. The reaction takes place completely in 3 hours Output (defined p is HPLC): 56%.

Example 10

Synthesis of 5'-O-DMTr-dGiBu-P(O)-dGiBu-3'-O-phosphoramidite using methylimidazole of triptoreline

106 mg of 5'-O-DMTr-dGiBu-P(O)-dGiBu-3'-HE (0.10 mmol, 1.0 EQ.) and 30 mg of metronidazole of triptoreline (0.15 mmol, 1.5 equiv.) dissolved in 10 ml of acetone, add 500 mg of molecular sieves 3E. After 30 min 34 ál 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (0.11 mmol, 1.1 equiv.) add to the mix the solution. The reaction takes place completely in 4 hours Output (determined by HPLC): 55%.

Example 11

Synthesis of 5'-O-DMTr-T-P(S)-dCBz-P(S)-T-P(S)-dCBz-P(S)-dCBz-P(S)-dCBz-3'-O-phosphoramidite using methylimidazole of triptoreline

10 mg of 5'-O-DMTr-T-P(S)-dCBz-P(S)-T-P(S)-dCBz-P(S)-dCBz-P(S)-dCBz-3'-HE (3.6 mmol, 1.0 EQ.) and 1.4 mg of metronidazole of triptoreline (7.2 mmol, 2.0 EQ.) dissolved in 0.5 ml of acetone and 0.5 ml of acetonitrile, add 50 mg of molecular sieves 3E. After 30 min of 5.8 ál 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (18,1 mmol, 5.0 equiv.) add to the mix the solution. The reaction takes place completely in 5 hours. Output (determined by HPLC): 71%.

Example 12

Synthesis of 5'-O-DMTr-dT-3'-O-phosphoramidite by generating in situ N-methylimidazole of triptoreline

to 1.00 g of 5'-O-DMTr-dT-3'-Oh (of 1.84 mmol, 1.0 EQ.) dissolved in 2 ml dichloromethane and 2 ml ACET is on. Add 300 mg of N-methylimidazole (3,68 mmol, 291 μl, 2.0 EQ.) and 665 mg 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (2.21 mmol, 700 μl, 1.2 EQ.) with the subsequent addition of 1.00 g of molecular sieves 3E. To this stirred suspension is added dropwise 230 mg triperoxonane acid (2.02 mmol, 159 μl, 1.1 EQ) in 1 ml dichloromethane. The reaction takes place completely in 3 hours Output (determined by HPLC): 99%.

Example 13

Synthesis of 5'-O-DMTr-dGiBu-3'-O-phosphoramidite by generating in situ N-methylimidazole of triptoreline

to 1.00 g of 5'-O-DMTr-dGiBu-3'-HE (1.56 mmol, 1.0 EQ.) dissolved in 2 ml dichloromethane and 2 ml of acetone. Add 255 mg of N-methylimidazole (3.11 mmol, 247 μl, 2.0 EQ.) and 563 mg 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (1,87 mmol, 593 μl, 1.2 EQ.) with the subsequent addition of 1.00 g of molecular sieves 3E. To this stirred suspension is added dropwise 195 mg triperoxonane acid (1,72 mmol, 135 μl, 1.1 EQ.) in 1 ml of dichloromethane. The reaction takes place completely in 5 hours. Output (determined by HPLC): 88%.

Example 14

Synthesis of 5'-O-DMTr-dGiBu-3'-O-phosphoramidite using a mixtureN-methylimidazole of triptoreline-N-methylimidazole

to 1.00 g of 5'-O-DMTr-dGiBu-3'-HE (1.56 mmol, 1.0 EQ.) dissolved in 2 ml dichloromethane and 2 ml of acetone. Add a 2.00 g of molecular sieves 3E, 367 mg of N-methylimidazole of triptoreline (1,87 mm is l, 1.2 EQ.) and 383 mg of N-methylimidazole (4,68 mmol, 371 μl, 3.0 EQ) followed by the addition 563 mg 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (1,87 mmol, 593 μl, 1.2 EQ.). The reaction takes place completely in 20 minutes Yield (determined by HPLC): 90%.

Example 15

Synthesis of 5'-O-DMTr-dCBz-3'-O-phosphoramidite using a mixture of N-methylimidazole of triptoreline-N-methylimidazole

to 1.00 g of 5'-O-DMTr-dGiBu-3'-HE (1.56 mmol, 1.0 EQ.) dissolved in 2 ml dichloromethane and 2 ml of acetone. Add a 2.00 g of molecular sieves 3E, 367 mg of N-methylimidazole of triptoreline (1,87 mmol, 1.2 EQ.) and 383 mg of N-methylimidazole (4,68 mmol, 371 μl, 3.0 EQ) followed by the addition 563 mg 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (1,87 mmol, 593 μl, 1.2 EQ.). The reaction takes place completely in 20 minutes Yield (determined by HPLC): 90%.

Example 16

Synthesis of 5'-O-DMTr-dCBz-P(O)-dABz-3'-O-phosphoramidite by the generation in situ of methylimidazole of triptoreline

100 mg of 5'-O-DMTr-dCBz-P(O)-dABz-3'-HE (90,7 µmol, 1.0 EQ.) dissolved in 200 µl of dichloromethane and 200 μl of acetone. Add 15 mg of N-methylimidazole (180 μmol, 14 μl, 2.0 EQ.) and 54.6 mg 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (181 mmol, 57 μl, 2.0 EQ.) with the subsequent addition of 100 mg of molecular sieves 3E. To this stirred suspension is added dropwise 100 μl of 1M solution trithorax the red acid in dichloromethane. The reaction takes place completely in 30 minutes Yield (determined by HPLC): 90%.

Example 17

Synthesis of 5'-O-phosphoramidite-dT-P(O)-dGiBu-P(O)-dGiBu-3'-O-Lev using N-methylimidazole of triptoreline

2.0 g of 5'-dT-P(O)-dGiBu-P(O)-dGiBu-3'-OLev (1.6 mmol, 1.0 EQ.) dissolved in 80 ml of acetone, add 500 mg of metronidazole of triptoreline (2.5 mmol, 1,56 EQ.) and 4.0 g of molecular sieves 3E. Add 2.76 ml 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (2,62 g, 8,7 mmol, 5 EQ.) and after 30 min of stirring phosphoramidic precipitated by adding 300 ml of n-heptane. Output (determined by HPLC): 72%.

Example 18

Synthesis of 5'-O-phosphoramidite-dCBz-P(O)-dABz-3'-O-Lev using N-methylimidazole of triptoreline

1.0 g of 5'-dCBz-P(O)-dABz-3'-OLev (1.1 mmol, 1.0 EQ.) and 326 mg of metronidazole of triptoreline (of 1.66 mmol, 1.5 EQ.) dissolved in 8 ml of acetone and add 10 ml dichloromethane and 2.0 g of molecular sieves 3E. Add 700 ál of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (664 mg, 2.2 mmol, 2 EQ.) and after 1 h of stirring phosphoramidic precipitated by adding 50 ml of n-heptane. Output (determined by HPLC): 78%.

Example 19

Synthesis of 5'-O-phosphoramidite-T-P(O)-dCBz-P(O)-dCBz-P(O)-dCBz-3'-O-Levthe use of N-methylimidazole of triptoreline

20 mg of 5'-T-P(O)-dC -P(O)-dCBz-P(O)-dCBz-3'-OLev (11.6 mmol, 1.0 EQ.) and 4.3 mg N-methylimidazole of triptoreline (22 mmol, 1.9 EQ.) dissolved in 2 ml of acetone and add 40 mg of the molecular sieve 3E. Add 15 ál of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (14 mg, 47 mmol, 4 equiv.) and after 1 h of stirring phosphoramidic precipitated by adding 3 ml of n-heptane. Output (determined by HPLC): 86%.

Example 20

Synthesis of 5'-O-TBDPS-dT-3'-O-phosphoramidite using N-methylimidazole of triptoreline

510 mg of 5'-O-DMTr-dT-3'-OH (1.06 mmol, 1.0 EQ.) dissolved in 20 ml of acetone and added with stirring 251 mg N-methylimidazole of triptoreline (1,27 mmol, 1.2 equiv.) 1.0 g of molecular sieves 3RD and 383 mg of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (403 μl, of 1.27 mmol, 1.2 EQ.). The reaction takes place completely in 30 minutes Yield (determined by HPLC): 88%.

Example 21

Synthesis of 5'-O-TBDMS-dGiBu-3'-O-phosphoramidite using N-methylimidazole of triptoreline

1 mg 5'-O-TBDMS-dGiBu-3'-OH (2.21 mmol, 1.0 EQ.) dissolved in 20 ml of acetone and added with stirring 875 mg N-methylimidazole of triptoreline (4,42 mmol, 2 equiv.) 2.0 g of molecular sieves 3RD and 3.33 g of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (3.5 ml, 11 mmol, 5 EQ.). The reaction takes place completely in 30 minutes Yield (determined by HPLC): 88%.

1. The method of receiving phosphoramidite, including article is DIU:
interaction of hydroxyl-containing compounds containing fragment sugar hospitaleros agent in the presence of an activator having the formula I

where R is alkyl, cycloalkyl, aryl, aralkyl, heteroalkyl, heteroaryl,
R1, R2or H, or together form a 5-6-membered cycle,
X1, X2- independently or N, or CH,
Y is H or Si(R4)3where R4- alkyl, cycloalkyl, aryl, aralkyl, heteroalkyl, heteroaryl,
In - triptorelin, dichloracetate, mesyl, tosyl, o-chlorophenolate.

2. The method according to claim 1, where the activator has the formula selected from the group consisting of

Y defined in claim 1,
R represents methyl, phenyl or benzyl.

3. The method according to claim 1 or 2, where the hydroxyl-containing compound is a nucleoside or an oligomer, which is a subclass.

4. The method according to claim 1 or 2, where the hydroxyl-containing compound is a 5'-O-protected nucleoside having a 3'-hydroxyl group or a 3'-O-protected nucleoside having a 5'-hydroxyl group.

5. The method according to claim 1 or 2, where the activator receive in situ and used without purification.

6. The method according to claim 1 or 2, where the reaction occurs in the presence of a mixture of an activator having the formula I, and the corresponding bases of the formula VIII

where R1, R2X1 X2and R is defined in claim 1.

7. The method according to claim 7, where the corresponding base is brought into contact with the hydroxyl-containing compound and hospitaleros agent and add the acid H+B-.

8. The method according to claim 1 or 2, where hospitalise agent has the formula II

where Z represents a leaving group, a R1and R2independently are secondary amino groups or halogen atoms.

9. The method of claim 8, where hospitalise agent is a 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite.

10. The method according to claim 1 or 2, where the reaction occurs in the presence of acetone.

11. The use of the activator having the formula I

where R is alkyl, cycloalkyl, aryl, aralkyl, heteroalkyl, heteroaryl,
R1, R2or H, or together form a 5-6-membered cycle,
X1, X2- independently or N, or CH,
Y is H or Si(R4)3where R4- alkyl, cycloalkyl, aryl, aralkyl, heteroalkyl, heteroaryl,
In - triptorelin, dichloracetate, mesyl, tosyl, o-chlorophenolate,
as activator for fosfaurilirovania hydroxyl-containing compounds containing fragment sugar, hospitaleros agent for receipt of phosphoramidite.

12. The application of item 11 to the synthesis of phosphoramidite adenosine, phosphoramidite cytosine, phosphoramidite guanosine and f is storemedia uracil, phosphoramidite deoxyadenosine, phosphoramidite of deoxyguanosine, phosphoramidite of deoxythymidine, phosphoramidite deoxycytidine and phosphoramidite oligonucleotides having the formula Xnwhere each X is chosen from A, dA, C, dC, G, dG, U, dT, and n=2-8, preferably 2-6, and their derivatives containing protective groups.

13. A mixture of activator having the formula I

where R is alkyl, cycloalkyl, aryl, aralkyl, heteroalkyl, heteroaryl,
R1, R2or H, or together form a 5-6-membered cycle,
X1, X2- independently or N, or CH,
Y is H or Si(R4)3where R4- alkyl, cycloalkyl, aryl, aralkyl, heteroalkyl, heteroaryl,
In - triptorelin, dichloracetate, mesyl, tosyl, o-chlorophenolate,
with the additive having the formula VIII

where R, R1, R2X1X2defined as for formula I, or pyridine at a molar ratio of from 1:1 to 1:10 (mol:mol).

14. The use of the mixture according to item 13 as activator for fosfaurilirovania hydroxyl-containing compounds containing fragment sugar, hospitaleros agent for receipt of phosphoramidite.

15. Apply the mixture on 14 for the synthesis of phosphoramidite adenosine, phosphoramidite cytosine, phosphoramidite guanosine and phosphoramidite uracil, phosphoramidite deoxyadenosine, phosphoramidite of desak is guanosine, phosphoramidite of deoxythymidine, phosphoramidite deoxycytidine and phosphoramidite oligonucleotides having the formula Xnwhere each X is chosen from A, dA, C, dC, G, dG, U, dT, and n=2-8, preferably 2-6, and their derivatives containing protective groups.

16. The use of ketones having the formula Rx-C(=O)-Rywhere Rxand Ryrepresent independently1-C6alkyl or together form cycloalkyl, as a reaction medium or co-solvent in the synthesis of phosphoramidites the method according to claims 1 to 10.

17. The application of article 16, where the ketone is an acetone, butanone, pentanone, hexanone, cyclohexanone or a mixture.



 

Same patents:

FIELD: medicine.

SUBSTANCE: invention describes nucleotide sequence (dwg.2), coding immunogenic polypeptide LcrV(G113), serving the base for construction of recombinant plasmid DNA pETV-I-3455, with the size 6538 bp, which codes immunogenic polypeptide LcrV(G113). Plasmid consists of plasmid pBR322 replicon, β-lactamase gene, determining resistance to ampicillin, T7-promoter, 1ac-operator, f1-replicon and DNA fragment, flanked by the sites for restrictases Ndel and Hindlll, coding synthesis of protein LcrV(G113), which starts from initiating codon ATG. Described is recombinant strain of bacteria E. coli BL21 (DE3)/pETV-I-3455 - producer of immunogenic polypeptide LcrV(G113) with amino acid sequence, represented on dwg.3, where tryptophan in position 113 (W113) is substituted with glycin. Described is method of obtaining said polypeptide by cultivation of strain E. coli BL21(DE3)/pETV-I-3455. Cells are destroyed in buffer solution by ultrasound and polypeptide is isolated successively by gel-permeation chromatography with application of carrier TSK HW-40, anion-exchanging and hydrophobic chromatography.

EFFECT: invention makes it possible to obtain product with high immunogenic and protective activity.

5 cl, 10 dwg, 2 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: what is offered is a method for producing high-polymer yeast RNA of used beer yeast. Used beer yeast concentrated by centrifugation is suspended in an aqueous solution of oleic acid titrated by alkali to pH 7-8. The suspension is kept at 98-102°C for 40-60 minutes. The hot lysate is settled for 20-24 h at room temperature. A supernatant is poured out with using a siphon. Then NaCl is added to the poured out liquid to the concentration 2-3 M. The suspension is kept for 20-96 h at room temperature. Then it is centrifuged without cooling in a low-speed centrifuge. The prepared cakes are consistently washed out in the NaCl solution 2-3 M and 92-96% ethanol by resuspension at room temperature and low-speed centrifugation.

EFFECT: invention promotes spread-out of the raw-material base and simplification of the high-polymer RNA technology.

1 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, and concerns mediated PHKi inhibition of RHO-kinase for treating ophthalmic disorders. The substance of the invention involves RNA-interference for mRNA Rho-kinase expression inhibition for treating patients with ophthalmic disorders, especially for treating intraocular pressure, eye hypertension and glaucoma The mRNA Rho-kinase targets include gene ROCK 1 mRNA.

EFFECT: creation of the agent exhibiting improved properties.

56 cl, 2 ex, 4 dwg, 1 tbl

FIELD: chemistry.

SUBSTANCE: disclosed is a preparation of high-polymer RNA from baker's yeast. The high-polymer RNA is extracted using sodium dodecyl sulphate. RNA is salted out with sodium chloride. Washing is carried out at low centrifuging rate of 1000 - 3000 g for 10 minutes without cooling.

EFFECT: obtained preparation is free from impurity association of RNA with proteins and polysaccharide and has narrow-directed stimulation of hemapoiesis.

1 dwg

FIELD: medicine.

SUBSTANCE: invention represents antibodies having an anti-MN-protein specific antigen linkage site, combinations and methods of application of such antibodies.

EFFECT: invention can be effectively used in treating and diagnosing MN-related disorders.

13 cl, 49 dwg, 4 tbl, 21 ex

FIELD: medicine.

SUBSTANCE: invention refers to DNA coding a modified antibody capable to identify and cross-link a TPO receptor, and containing two or more V-areas of the N-chain and two or more V-areas of the L-chain of an initial antibody connected directly or through a covalent or noncovalent linker of a smaller size in comparison with the initial antibody. DNA includes two or more DNA-sequences coding the V-areas of the L-chain and N-chains; DNA coding at least one of the V-areas of the L-chain and/or the N-chain includes specific nucleotide sequences presented in the description. The invention discloses DNA coding a compound showing an equal or more agonist action (ED50) compared with thrombopoietin (TPO); the compound can represent the modified antibody, the whole antibody or F(ab ')2 capable to identify specifically and cross-link the TPO receptor. Also, the invention relates to a vector containing specified DNA, an animal cell and a microorganism which contain DNA or the vector and produce the modified antibody or the compound with the TPO agonist activity. The invention describes methods of producing the modified antibody - TPO agonist and the compounds which involve cultivation of the transformed animal cell or microorganism containing DNA of the invention.

EFFECT: products of the invention can be applied as signal transduction TRO-agonists, and can be used as a prophylactic or therapeutic agent in blood disorders associated with connected with thrombocyte count reduction, thrombopenia accompanying chemotherapy in cancer or leukaemia.

33 cl, 61 dwg, 3 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: invention can be used for determining a functional status of primary signalling pathways of a cell. Substance of the invention are lentiviral reporter vector structures containing reporter markers. The invention allows predicting the tumour sensitivity to therapeutic exposures and developing an individual therapeutic approach.

EFFECT: creating the lentiviral reporter vector structure optimal for determining the activity of transcriptional factors reflecting the functional status of signalling pathways of the cell.

6 cl, 16 dwg

FIELD: medicine.

SUBSTANCE: invention refers to a synthetic oligonucleotide kit for identifying DNA of human monocytic ehrlichiosis (HME) agent - pathogenic representatives of Ehrlichia genus by a polymerase chain reaction. The offered invention can be used for the diagnostic purposes for detecting monocytic ehrlichiosis Ehrlichia spp. by the real-time polymerase chain reaction. Said kit includes primers as follows: 5'- GGG GAA AGA TTT ATC GCT ATT AG -3', 5'- CGG CAT AGC TGG ATC AGG CT -3' and a sample: (BHQl)-5'- CCC ACT GCT GCC (FdT)CC CGT AGG AGT CTG G - 3'P, where BHQ1 means a dark fluorescence killer attached to 5'-terminal nucleotide, while FdT is a fluorescent dye FAM attached to nucleotide T.

EFFECT: invention allows reliable identification of Ehrlichia representatives in the biological material.

1 ex

FIELD: medicine.

SUBSTANCE: invention is intended for inhibiting reproduction of influenza A viruses in infected eukaryotic cells by means of virus-specific catalytically active oligodeoxyribonucleotide (deoxyribozyme). Method is realised by obtaining deoxyribozyme, consisting of nucleotide sequence: 5'-GAAATAAGAGGCTAGCTACAACGACCTTCATTA, intended for inhibiting reproduction of influenza A viruses.

EFFECT: extension of spectrum of anti-viral action of deoxyribozymes and increase of their anti-viral activity.

3 dwg, 1 tbl, 2 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biochemistry, particularly to a set of primers for amplifying the L1 gene of the human papilloma virus (HPV), a set for detecting the human papilloma virus (HPV) genotype and methods of detecting human papilloma virus (HPV) genotypes. The method involves primary PCR amplification of the HPV L1 gene in an analysed sample using a set of direct and reverse primers which are specific for the L1 gene section. Further, secondary PCR amplification of the product of primary PCR amplification of the L1 gene is carried out using a reverse primer to obtain a biotin-labelled single-stranded L1 gene. The product of secondary PCR amplification and biotin-labelled single-stranded L1 gene undergo a hybridisation reaction with one or more probes for detecting the HPV genotype or a probe for detecting the HPV genotype. Further, the product of the hybridisation reaction reacts with phycoerythrin, bonded with streptavidin. The level of fluorescent substance is measured and probes are identified in the product of the hybridisation reaction to identify the HPV genotype. Further, the HPV genotype is determined, as well as the level of its presence in accordance with probes and the level of the fluorescent substance.

EFFECT: invention provides a method for detecting HPV in a sample with high sensitivity, sufficient for detecting extremely small quantities of HPV in the sample.

8 cl, 2 dwg, 1 tbl, 4 ex

FIELD: biotechnology.

SUBSTANCE: nucleic acids are extracted from biomass in the presence of hydrogen peroxide in the low concentration as retaining substance. Obtained extract is treated with proteolytic enzyme and RNA is precipitated with acid in the presence of calcium salt. Purification of product is carried out with aqueous solutions of acetone and calcium and sodium chlorides followed by purification of sodium nucleate with an aqueous-acetone solution in the presence of activated carbon. Invention provides elevating yield of the end product and to improve its quality. Invention can be used for preparing sodium nucleate from baking yeast.

EFFECT: improved preparing method.

1 ex

FIELD: biotechnology, medicine, proteins.

SUBSTANCE: invention describes new polypeptide in isolated form relating to subfamily of superfamily human immunoglobulins (Ig-Sf). This polypeptide shows at least 70% of homology level with amino acid sequence of murine molecules CRAM-1 or CRAM-2 regulated by the confluence of adhesive (figures 3, 6 are represented in the claim). Also, invention relates to antibodies showing specificity with respect to the polypeptide. Antibodies and soluble polypeptide can be used for treatment of inflammation and tumors. Invention describes polynucleotide or oligonucleotide encoding the full-size polypeptide or its moiety and represents primer, probe, anti-sense RNA and shows the nucleotide sequence that is identical conceptually with human CRAM-1. Invention provides preparing new adhesive proteins from superfamily Ig-Sf that are regulated at the transcription level in endothelium by effect of tumors. Invention can be used for treatment of different diseases, in particular, inflammatory responses.

EFFECT: valuable medicinal properties of polypeptide.

19 cl, 33 dwg, 1 ex

FIELD: biochemistry.

SUBSTANCE: the present innovation deals with an anti-sense oligonucleotide or one of its derivatives which can inhibit expression of human eg5 protein being relative to kinesin of motor proteins. The oligonucleotide has got a sequence being correspondent to that of nucleic acid coding certain part of human eg5. This innovation deals with the way to obtain the above-mentioned oligonucleotides, pharmaceutical composition for inhibiting human eg5 and its application. Advantage of the innovation deals with developing e new preparation to be applied for inhibiting cell proliferation.

EFFECT: higher efficiency of inhibition.

11 cl, 1 dwg, 2 ex, 3 tbl

FIELD: medicine, pharmaceutics.

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EFFECT: increased anti-heparin activity.

FIELD: genetic engineering, medicine.

SUBSTANCE: invention relates to T-cell receptor sequence being detected in patients with extended sclerosis and is useful in diagnosis and therapy. Oligonucleotide including sequence which represents or is derived from 5'-CTAGGGCGGGCGGGACTCACCTAC-3' or nucleotide sequence being fully complementary thereto. Oligonucleotide together with nuclear acid including nearly 15-30 oligonucleotides, which doesn't comprise oligonucleotide sequence and presents in region from Vβ to Jβ of Vβ13.1 gene in T-cell Vβ13.1-subgroup, wherein oligonucleotide and nuclear acid sequences don't present in the same chain of pair sequences of Vβ13.1 gene, is used in Vβ13.1 gene part amplification. In method for detection of LGRAGLTY motive, which is present in T-cell receptors of T-cell Vβ13.1-subgroup, oligonucleotide is used in combination with labeling particle. Once LGRAGLTY motive is detected, development monitoring and treatment are carried out by removing of LGRAGLTY motive-containing peptide.

EFFECT: simplified methods for detection of LGRAGLTY motive in T-cell receptors and treatment of patients with extended sclerosis.

21 cl, 7 dwg, 3 tbl, 3 ex

FIELD: genetic engineering, medicine.

SUBSTANCE: invention relates to T-cell receptor sequence being detected in patients with extended sclerosis and is useful in diagnosis and therapy. Oligonucleotide including sequence which represents or is derived from 5'-CTAGGGCGGGCGGGACTCACCTAC-3' or nucleotide sequence being fully complementary thereto. Oligonucleotide together with nuclear acid including nearly 15-30 oligonucleotides, which doesn't comprise oligonucleotide sequence and presents in region from Vβ to Jβ of Vβ13.1 gene in T-cell Vβ13.1-subgroup, wherein oligonucleotide and nuclear acid sequences don't present in the same chain of pair sequences of Vβ13.1 gene, is used in Vβ13.1 gene part amplification. In method for detection of LGRAGLTY motive, which is present in T-cell receptors of T-cell Vβ13.1-subgroup, oligonucleotide is used in combination with labeling particle. Once LGRAGLTY motive is detected, development monitoring and treatment are carried out by removing of LGRAGLTY motive-containing peptide.

EFFECT: simplified methods for detection of LGRAGLTY motive in T-cell receptors and treatment of patients with extended sclerosis.

21 cl, 7 dwg, 3 tbl, 3 ex

FIELD: medicine, genetics, biochemistry.

SUBSTANCE: invention relates to new NOS-variants or mutants that comprise structural modifications in site Akt-dependent phosphorylation. Modified NOS-proteins or peptides, in particular, human proteins or eNOS-peptides having change of amino acid residue corresponding to S/T in motif of the consensus-sequence RXRXXS/T of NOS-polypeptide of wild type and nucleic acid molecules encoding thereof can be used in genetic therapy and proteins and NOS-peptides can be used in screening methods of agents modulating activity of NOS. The advantage of invention involves the creature of new NOS-variants or mutants that can be used in genetic therapy.

EFFECT: valuable medicinal properties of mutants.

25 cl, 1 tbl, 9 dwg, 3 ex

FIELD: organic chemistry, biochemistry.

SUBSTANCE: invention relates to oligomer comprising at least one nucleoside analogue of L-ribo-CNA of the general formula (Ia) wherein X represents -O-; B represents nitrogen base; P means radical position in an internucleoside linkage followed by monomer or 5'-terminal hydroxy-group; P* means an internucleoside linkage with precede monomer or 3'-terminal hydroxy-group; R2* and R4* mean in common biradical -(CH2)0-1-O-(CH2)1-3-(CH2)0-1-S-(CH2)1-3- or -(CH2)0-1-NR-(CH2)1-3- wherein R means hydrogen atom, alkyl or acyl; R1*, R2, R3*, R5 and R5* mean hydrogen atom. Also, invention proposes nucleoside analogues used in preparing oligomers. Proposed oligomers elicit the enhanced affinity to complementary nucleic acids and can be used as a tool in molecular-biological investigations and as antisense, antigen agents of agents activating genes.

EFFECT: valuable properties of analogues.

15 cl, 3 tbl, 4 dwg, 17 ex

FIELD: molecular biology, medicine, pharmaceutical industry.

SUBSTANCE: method for detecting analyzed DNA sequence involves DNA hybridization with probes and visualization the prepared product wherein probes represent oligonucleotides with length of nucleotide sequence 12-30 nucleotides showing complementary to site of the same size in analyzed DNA that are modified with insertions based on alkyldiols or ethylene glycols. Applying the proposed method provides obtaining more reliable and selective results in detecting analyzed DNA sequences.

EFFECT: improved detecting method of DNA sequence.

11 cl, 12 dwg, 13 ex

FIELD: biotechnology.

SUBSTANCE: invention relates to polynucleotide encoding zwal gene product containing polynucleotide sequence selected from group including a) polynucleotide encoding polupeptide with amino acid sequence with at least 90 % identity to amino acid sequence represented in SEQ ID NO:2; b) polynucleotide which is complementary to polynucleotides from a), as well as primer representing polynucleotide containing at least 15 sequential base pairs of abovementioned polynucleotide.

EFFECT: new zwal gene encoding ionic zwal product.

6 cl, 1 dwg, 1 tbl, 5 ex

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