Patent Application: US-55535104-A

Abstract:
new class of metallated nucleoside and nucleotide conjugates containing metallacarborane cage is provided , also oligonucleotides containing metallacarborane modification are disclosed . nucleosides , nucleotides and oligonucleotides containing metallacarborane cage can be used among others as modified primers in amplification of rna and dna , antisense drugs , boron carriers for bnct , radiopharmaceuticals bearing a range of isotopes useful in different types of radiotherapy , molecular probes , elements of biosensors , materials for nanotechnology and others .

Description:
the term alkyl , as used herein , unless otherwise specified , refers to a saturated straight , branched , or cyclic , primary , secondary , or tertiary hydrocarbon of c1 to c10 , and specifically includes methyl , ethyl , propyl , isopropyl , butyl , isobutyl , t - butyl , pentyl , cyclopentyl , isopentyl , neopentyl , hexyl , isohexyl , cyclohexyl , cyclohexylmethyl , 3 - methylpentyl , 2 , 2 - dimethylbutyl , and 2 , 3 - dimethylbutyl . the alkyl group can be optionally substituted with one or more moieties selected from the group consisting of hydroxyl , amino , alkylamino , arylamino , alkoxy , aryloxy , nitro , cyano , sulfonic acid , sulfate , phosphonic acid , phosphate , or phosphonate , either unprotected , or protected as necessary , as known to those skilled in the art , for example , as taught in greene , et al ., “ protective groups in organic synthesis ,” john wiley and sons , second edition , 1991 . the term lower alkyl , as used herein , and unless otherwise specified , refers to a c1 to c4 saturated straight or branched alkyl group . the term alkylamino or arylamino refers to an amino group that has one or two alkyl or aryl substituents , respectively . the term “ protected ” as used herein and unless otherwise defined refers to a group that is added to an oxygen , nitrogen , or phosphorus atom to prevent its further reaction or for other purposes . a wide variety of oxygen and nitrogen protecting groups are known to those skilled in the art of organic synthesis . the term aryl , as used herein , and unless otherwise specified , refers to phenyl , biphenyl , or naphthyl , and preferably phenyl . the aryl group can be optionally substituted with one or more moieties selected from the group consisting of hydroxyl , amino , alkylamino , arylamino , alkoxy , aryloxy , nitro , cyano , sulfonic acid , sulfate , phosphonic acid , phosphate , or phosphonate , either unprotected , or protected as necessary , as known to those skilled in the art , for example , as taught in greene , et al ., “ protective groups in organic synthesis ,” john wiley and sons , second edition , 1991 . the term alkaryl or alkylaryl refers to an alkyl group with an aryl substituent . the term aralkyl or arylalkyl refers to an aryl group with an alkyl substituent . the term halo , as used herein , includes chloro , bromo , iodo , and fluoro . the term purine or pyrimidine base includes , but is not limited to , adenine , n6 - alkyulpurines , n6 - acylpurines ( wherein acyl is c ( o ) ( alkyl , aryl , alkylaryl , or arylalkyl ), n6 - benzylpurine , n6 - halopurine , n6 - vinylpurine , n6 - acetylenic purine , n6 - acyl purine , n6 - hydroxyalkyl purine , n6 - thioalkyl purine , n2 - alkylpurines , n2 - alkyl - 6 - thiopurines , thymine , cytosine , 6 - azapyrimidine , 2 - and / or 4 - thiopyrmidine , uracil , c5 - alkylpyrimidines , c5 - benzylpyrimidines , c5 - halopyrimidines , c5 - vinlypyrimidine , c5 - acetylenic pyrimidine , c5 - acyl pyrimidine , c5 - hydroxyalkyl purine , c5 amidopyrimidine , c5 - cyanopyrimidine , c5 - nitropyrimidine , c5 - aminopyrimidine , n2 - alkylpurines , n2 - alkyl - 6 - thipourines , 5 - azacylidinyl , 5 - azauracilyl , triazolopyridinyl , imidazolopyridinyl , pyrrolopyrimidinyl , pyrazolopyrimidinyl . functional oxygen and nitrogen groups on the base can be protected as necessary or desired . suitable protecting groups are well known to those skilled in the art , and include trimethylsilyl , dimethylhexylsilyl , t - butyldimethylsilyl , and t - butyldiphenylsilyl , trityl , alkyl groups , acyl groups such as acetyl and propionyl , methylsulfonyl , and p - toluylsulfonyl . the term heteroaryl or heteroaromatic , as used herein , refers to an aromatic moiety that includes at least one sulfur , oxygen , or nitrogen in the aromatic ring . nonlimiting examples are ffiryl , pyridyl , pyrimidyl , thienyl , isothiazolyl , imidazolyl , tetrazolyl , pyrazinyl , benzofuranyl , benzothiophenyl , quinolyl , isoquinolyl , benzothienyl , isobenzofuryl , pyrazolyl , indolyl , isoindolyl , benzimidazolyl , purinyl , carbazolyl , oxazolyl , thiazolyl , isothiazolyl , 1 , 2 , 4 - thiadiazolyl , isooxazolyl , pyrrolyl , quinazolinyl , pyridazinyl , pyrazinyl , cinnolinyl , phthalazinyl , quinoxalinyl , xanthinyl , hypoxanthinyl , and pteridinyl . functional oxygen and nitrogen groups on the heterocyclic base can be protected as necessary or desired . suitable protecting groups are well known to those skilled in the art , and include trimethylsilyl , dimethylhexylsilyl , t - butyldimethylsilyl , and t - butyldiphenylsilyl , trityl or substituted trityl , alkyl groups , acyl groups such as acetyl and propionyl , methylsulfonyl , and p - toluylsulfonyl . the term alkenyl , as referred to herein , and unless otherwise specified , refers to a straight , branched , hydrocarbon of c2 to c10 with at least one double bond . the term acyl refers to moiety of the formula — c ( o ) r ′, wherein r ′ is alkyl ; alkoxyalkyl including methoxymethyl ; arylalkyl including benzyl ; aryloxyalkyl such as phenoxymethyl ; aryl including phenyl optionally substituted with halogen , c1 to c4 alkyl or c1 to c4 alkoxy , or the residue of an amino acid . the term oligonucleotide refers to an oligomer of thirty - five or less nucleotides linked through their 3 ′ and 5 ′- hydroxyl or 2 ′- and 5 ′- hydroxyl groups . the term “ metallacarboranes ”, as used herein , refers to compounds of metallocene type complexes consisting at least one carborane cage ligand and one or more metal atoms . for a review of metallacarborane chemistry see grimes , r . n ., 2000 . metallacarboranes in the new millennium , coord . chem . rev ., 200 - 202 , 773 - 811 ; saxena , a . k ., hosmane , n . s ., 1993 . recent advances in the chemistry of carborane metal complexes incorporating d - and f - block elements . chem . rev ., 93 , 1081 - 1124 ; grimes , r . n ., transition metal metallacarboranes in comprehensive organometallic chemistry , 1995 , vol . 1 , housecroft c . e ., ed ., pergamon , n . y ., 373 - 430 . derivative ( of the nucleoside , nucleotide , oligonucleotide , protein , antibodies and other biological molecules ) is a chemically or physically changed form of the mother molecule ( substrate ). dna hybridization technology — the technologies and their applications based on specific formation of double stranded nucleic acid structure due to specific nucleic bases recognition according to watson - crick rule . dna microarrays — terminologies that have been used in the literature to describe this technology include , but not limited to : biochip , dna chip , dna microarray , and gene array . affymetrix inc . owns a registered trademark , genechip ®, which refers to its high density , oligonucleotide - based dna arrays . however , in some articles appeared in professional journals , popular magazines , and the www the term “ gene chip ( s )” has been used as a general terminology that refers to the microarray technology . base - pairing ( i . e ., a - t and g - c for dna ; a - u and g - c for rna ) or hybridization is the underlining principle of dna microarray . an array is an orderly arrangement of samples . it provides a medium for matching known and unknown dna samples based on base - pairing rules and automating the process of identifying the unknowns . in general , arrays are described as macroarrays or microarrays , the difference being the size of the sample spots . macroarrays contain sample spot sizes of about 300 microns or larger and can be easily imaged by existing gel and blot scanners . the sample spot sizes in microarray are typically less than 200 microns in diameter and these arrays usually contains thousands of spots . dna sensor , is an immobilized dna recognition element ( receptor ) coupled to a transducer unit and an electronic amplifier . dependent on the kind of interaction between the analyze ( s ) and the receptor ( r ) one differ entities between : affinity sensors ( affinity to the substrate ; s + r “& lt ;−−& gt ;” rs ), metabolism sensors ( use of the substrate ; s + r “& lt ;−−& gt ;” rs “& lt ;−−& gt ;” p + r ). electrochemical dna sensors , the device detecting hybridization process between target nucleic acid and nucleic acid probe . the dna sensor comprises a nucleic acid recognition layer and a signal transducer . the signal transducer determine that that hybridization occurred on the base of the electrochemical process triggered by the hybridization and converts this into electronic signal nanotechnology , the development and use of devices that have a size of only a few nanometres . research has been carried out into very small components , many of which depend on quantum effects and may involve movement of a very small number of electrons in their action . such devices would act faster than larger components . considerable interest has been shown in the production of structures on a molecular level by suitable sequences of chemical reactions or lithographic techniques . it is also possible to manipulate individual atoms on surfaces using a variant of the atomic force microscope to make , for example , high density data storage devices . nucleoside is defined as a compound consisting a five - carbon sugar molecule ( a pentose , ribose or deoxyribose ) and an organic base , purine or pyrimidine . the procedure was performed under positive pressure of argon . 5 ′- o - monomethoxytrityl - 3 ′- o - acetylthymidyne ( 22 ) ( 0 . 9 g , 1 . 6 mmol ) and 8 - dioxane - cosan ( 2 , 1 . 4 g , 3 . 3 mmol ) were mixed together then dried under high vacuum over p 2 0 5 for 24 h , next nah ( 60 % suspension in mineral oil , 80 mg , 3 . 3 mmole ) was added followed by anhydrous toluene ( 18 ml ). the reaction mixture was stirred at 70 ° c . in oil bath . after 8 h an excess of nah was removed by centrifugation and supernatant added dropwise into hexane ( 135 ml ). the resultant precipitate was separated by centrifugation and the sediment was dried under vacuum yielding 2 . 4 g of crude 24 as a mixture of 3 - n and 4 - o isomers ( 24a and 24b , respectively ). crude 24 was purified by silica gel column chromatography ( 70g silica gel , 230 - 400 mesh ) using 12 % ch 3 oh in chcl 3 , containing 1 % et 3 n as eluting solvent system . the yield of 24a was 221 mg and 24b was 450 mg . 24a : tlc ( ch 3 cn / chcl 3 , 1 : 2 ): r f 0 . 14 ; uv ( ch 3 cn ): λ min 227 . 46 , 249 . 18 , 287 . 30 nm , λ max 234 . 43 , 261 . 89 , 313 . 93 mn ; 1 h - nmr ( c 6 d 6 ): 1 . 8 - 3 . 5 ( bm , 21h , bh - cosan ), 1 . 58 ( s , 3h , ch 3 co ), 1 . 71 ( s , 3h , ch 3 - 5 ), 2 . 54 ( m , 2h - 2 ′), 2 . 95 - 3 . 08 ( m , 4h , 4 × ch - cosan ), 3 . 28 - 3 . 41 ( bs , 4h , 2 × och 2 ), 3 . 43 ( s , 3h , ch 3 o ), 3 . 46 - 3 . 52 ( q , 2h - 5 ′, 5 ″), 3 . 92 ( bs , 2h , och 2 ), 3 , 96 ( s , 1h - 4 ′), 4 . 48 - 4 . 59 ( m , 2h , och 2 ), 5 . 36 ( s , 1h - 3 ′), 6 . 31 ( s , — h arom . in 4 - ch1h - 1 ′), 6 . 87 ( d , 2h , 3 oph ), 7 . 28 - 7 . 60 ( m , 12h , h arom . in mmtr ), 8 . 11 ( s , 1h - 6 ); 13 c - nmr ( c 6 d 6 ): 13 . 62 ( ch 3 - 5 ), 20 . 99 ( ch 3 in ch 3 co ), 39 . 76 ( c - 2 ′), 48 . 10 , 51 . 94 ( c - cosan ), 55 . 72 ( ch 3 o ), 64 . 61 ( c - 5 ′), 68 . 73 , 73 . 48 ( och 2 ), 75 . 67 ( c - 3 ′), 86 . 41 ( c - 4 ′), 87 . 46 ( c - 1 ′), 88 . 74 ( c - methylidene in mmtr ), 114 . 65 , 129 . 23 , 131 . 40 , 144 . 85 , 145 . 06 ( mmtr ), 135 . 92 - c in 4 - ch ( c - 6 ), 156 . 91 ( c - 2 ), 160 . 24 ( 3 oph ), 170 . 30 ( co in ch 3 co ), 176 . 38 ( c - 4 ); 11 b - nmr ( c 6 d 6 ); 30 . 50 - 32 . 00 ( bs , 18b ); fab - ms (- ve , nba ) 966 . 9 [ m - 1 ]. 24b : tlc ( ch 3 cn / chcl 3 , 1 : 2 ): r f 0 . 41 ; uv ( ch 3 cn ): λ min 252 . 87 nm , λ max 283 . 91 nm , 311 . 84 nm ; 1 h - nmr ( c 6 d 6 ): 1 . 50 - 4 . 00 ( bm , 21h , bh - cosan ), 1 . 48 ( s , 3h , ch 3 - 5 ), 1 . 62 ( s , 3h , ch 3 co ), 2 . 55 ( m , 2h - 2 ′), 2 . 77 - 3 . 07 ( m , 4h , 4 × ch — cosan ), 3 . 21 ( bs , 4h , 2 × och 2 ), 3 . 37 ( s , 3h , ch 3 o ), 3 . 45 - 3 . 51 ( q , 2h - 5 ′, 5 41 , j 5 ′ 5 ″ = 9 . 04 ), 3 . 88 ( bs , 2h , och 2 ), 4 . 08 ( s , 1h - 4 ′), 4 . 37 - 4 . 43 ( m , 2h , och 2 ), 5 . 43 ( s , 1h - 3 ′), 6 . 64 ( s , — h arom . in ch1h - 1 ′), 6 . 82 ( d , 2h , 3 oph ), 7 . 06 - 7 . 52 ( m , 12h , h arom . in mmtr ), 8 . 20 ( s , 1h - 6 ); 13 c - nmr ( c 6 d 6 ): 12 . 33 ( ch 3 - 5 ), 21 . 09 ( ch 3 - acetyl ), 40 . 22 ( c - 2 ′), 48 . 12 , 51 . 85 , 51 . 95 ( c - cosan ), 55 . 60 ( ch 3 o ), 64 . 43 ( c - 5 ′), 68 . 27 , 71 . 68 , 73 . 26 ( och 2 ), 75 . 53 ( c - 3 ′), 85 . 97 ( c - 4 ′), 87 . 75 ( c - 1 ′), 88 . 56 ( c - methylidene in mmtr ), 114 . 55 , 129 . 14 , 129 . 23 , 129 . 55 , 131 . 41 , 142 . 33 , 144 . 94 , 145 . 09 ( mmtr ), 135 . 95 ( c - 6 ), 158 . 93 ( c - 2 ), 160 . 21 ( c - 4 in 4 - ch 3 oph ), 170 . 58 ( c - 4 ), 172 . 52 ( ch 3 - acetyl ); 11 b - nmr ( c 6 d 6 ): 30 . 50 - 32 . 00 ( bs , 18b ); fab - ms (- ve , nba ) 966 , 9 [ m - 1 ]. analogously other metallacarboranes can be incorporated into purine or pyrimidine nucleic base and sugar residue of the nucleoside unit . ( 0 . 1 g , 0 . 11 mmol ) was dissolved in ch 3 cn ( 5 ml ) then to the resultant solution acetic acid was added ( 80 % ch 3 cooh , 10 ml ). after 2 . 5 h at room temperature the reaction went to the completion ( tlc control , solvent system chcl 3 / ch 3 oh , 8 : 2 ) and the solvents were evaporated under reduced pressure yielding crude 28 ( 0 . 18 g ). crude 28 was purified by silica gel column chromatography ( 2 g of silica gel , 230 - 400 mesh ) using 30 % ch 3 oh in chcl 3 as eluting solvent system . the yield of 28 was 40 mg ( 58 %). tlc ( chcl 3 / ch 3 oh , 8 : 2 ): r f 0 , 18 ; uv ( ch 3 cn ): λ min 237 , 93 , λ max 282 , 76 , λ max 310 , 67 ; 1 h - nmr ( cd 3 od ): 1 . 2 - 3 . 2 ( bm , 21h , bh - cosan ), 2 . 02 ( s , 3h , ch 3 - 5 ), 2 . 16 - 2 . 21 ( m 1h - 2 ′), 2 . 40 - 2 . 44 ( m , 1h - 2 ″), 3 . 60 - 3 . 65 ( m , 4h , 2 × och2 ), 3 . 73 - 3 . 76 ( dd , 1h - 5 ′), 3 . 82 - 3 . 85 ( m , 3h , 1h - 5 ″ and och 2 ), 3 . 96 ( q , 1h - 4 ′), 4 . 13 ( s , 2h , 2 × ch - cosan ), 4 . 38 - 4 . 39 ( m , 1h - 3 ′), 4 . 42 - 4 . 46 ( m , 1h , och 2 ), 4 . 52 - 4 . 56 ( m , 1h , och 2 ), 6 . 26 ( t , 1h - 1 ′, j 1 ′ 2 ′ = 6 . 36 ), 8 . 13 ( s , 1h - 6 ); 13 c - nmr ( cd 3 od ): 12 . 40 ( ch 3 - 5 ), 42 . 24 ( c - 2 ′), 48 . 04 ( c - cosan ), 55 . 15 ( c - cosan ), 62 . 54 ( c - 5 ′), 68 . 13 ( och 2 ), 69 . 83 ( och 2 ), 69 . 98 ( och 2 ), 71 . 69 ( c - 3 ′), 73 . 07 ( och 2 ), 87 . 97 ( c - 1 ′), 89 . 11 ( c - 4 ′), 142 . 08 ( c - 6 ), 158 . 20 ( c - 2 ), 172 . 16 ( c - 4 ); 11 b - nmr ( cd 3 od , h - decoupled ): 28 . 54 ( s ), 10 . 30 ( s ), 6 . 33 ( s ), 3 . 48 ( s ), 1 . 32 ( s ), from − 1 , 59 to − 2 , 19 ( d ), − 11 , 47 ( s ), − 14 , 68 ( s ); fab - ms (- ve , nba ) 652 , 6 [ m - 1 ]. method for the automated production of oligonucleotides are described below ( example 3 and 4 ). given the disclosure herein , one of ordinary skill in the art will know how to prepare a wide variety of oligonucleotides with metallacarborane - containing nucleotide monomers for a diverse range of applications , all of which are intended to fall within the scope of this invention . all procedures were performed under positive pressure of argon . 5 ′- o - monomethoxytrityl - 4 - o -( bisethoxy - 8 - cosan ) thymidine ( 25a ) ( 0 . 1 g , 0 . 11 mmol ) was dried under high vacuum over p 2 0s for not less than 12 h , then was dissolved in ch 2 cl 2 ( fresh distilled over cah 2 , 1 . 3 ml ) and next n , n - diisopropylethylamine ( 0 . 075 ml ) was added . to the resultant solution 2 - cyanoethyl - n , n - diisopropylchlorophosphoramidite was added drpowise ( 0 . 072 ml , 0 . 32 mmole ). after 4 h the reaction mixture was washed with h 2 o ( 3 × 5 ml ) then the organic layer was dried over mgso 4 and solvent evaporated . the crude product 7 was obtained as colorless oil ( 0 . 11 g ). crude 7 was purified by silica gel column chromatography ( 6 g of silica gel , 230 - 400 mesh ) using ch 3 cn / ch 2 cl 2 ( 1 : 3 ) as eluting solvent system . the yield of 7 was 78 mg ( 65 %). tlc ( ch 3 cn / ch 2 cl 2 , 1 : 3 ): r f 0 . 25 , uv ( anhydrous ch 3 cn ): λ min 255 . 2 nm , λ max 281 . 6 nm , λ max 311 . 3 nm ; 31 p - nmr ( c 6 d 6 ): 149 , 00 and 149 , 82 ( 1 : 1 ), fab - ms [- ve , nba ] 1177 [ m + 2 × na ] 4 - o -( diethyleneoxy - 8 - cosan ) thymidine ( bec t )- containing tetradecanucleotides 5 ′- d ( bec t gctggtttggctg )- 3 ′ ( 8 ) and unmodified oligonucleotide 5 ′- d ( cgctggt7tggctg )- 3 ′ ( 9 ) ( fig7 ). the natural oligonucleotide 9 and the modified oligonucleotide 8 were synthesized using a beckman oligo 1000 dna synthesizer . column loaded with controlled pore glass functionalized with 5 ′- o - dimethoxytrityl 2 ′- o - deoxyguanosine mol ) were used as solid support . suitable 5 ′- o - dimethoxytrityl - 2 ′- o - deoxynucleoside 3 ′-( n , n - diisopropyl - beta - cyanoethyl ) phosphor - amidites ( 9 , b = gua , cyt , thy ) were prepared as a 0 . 5 g / 10 ml solution in anhydrous acetonitrile . elongation of the oligomers with natural nucleotide was performed using a standard 0 . 2 pmol dna synthesis beta - cyanoethyl cycle without changes in condensation time . coupling of the 5 ′- terminal modified monomer 5 ′- o - monomethoxytrityl - 4 - o -( bisethoxy - 8 - cosan ) thymidine 3 ′- o -( n , n - diisopropyl - 2 - cyanoethyl ) phosphoramidite ( 26 ) as well as oxidation step were performed manually ( capping step was omitted ). after thirteen coupling cycles , detritylation and washing with acetonitrile the column was detached from the dna synthesizer and dried under high vacuum ( 5 min ). monomer 26 ( 20 mg , 0 . 02 mmol ) was dissolved in anhydrous acetonitrile ( 120 μl ) followed by addition of tetrazole ( 0 . 09 ml , 0 . 5 m , 0 . 045 mmol ). a solution of activated 26 was applied to the column and the coupling reaction was performed for 30 min . the column was washed with anhydrous acetonitrile ( 2 × 5 ml ) followed by drying under high vacuum . the oxidation step was performed using a tert - butyl hydroperoxide solution ( 0 . 5 m , 1 ml ) for 2 min . followed by washing with acetonitrile ( 1 × 5 ml ) and drying under high vacuum . oligonucleotides were then cleaved from the support by 1 h incubation with concentrated aqueous ammonia solution ( 30 %, 1 nml ) at room temperature then base deprotection was achieved by incubation of resultant solution at 50 ° c . for 2 h . the solution of crude 5 ′- o - monomethoxytrityl protected oligonucleotide 8 and 5 ′- o - dimethoxytrityl protected oligomer 9 was degassed with a stream of argon and evaporated to dryness under vacuum , then redissolved in water . resultant solution of crude 5 ′- o - protected oligonucleotides 8 and 9 having 33 and 64 a 260 optical density units , respectively , were purified using hplc c 18 reverse phase column ( rp - hplc ) using conditions as follows : 20 min from 0 % b to 100 % b , 5 min 100 % b , 5 min from 100 % b to 0 % b . fractions containing the desired product were collected , and the buffer was evaporated under vacuum . the residue was co - evaporated with 96 % ethyl alcohol to remove triethylammonium bicarbonate ( teab ), then detritylation was performed using a 80 % acetic acid ( 1 . 0 ml ) at room temperature for 20 min . next the acetic acid solution was evaporated to dryness under vacuum and the totally deprotected oligonucleotides were purified by rp - hplc using conditions as above . buffer a contained 0 . 1 m teab ( ph 7 . 0 ) in a mixture of acetonitrile and water ( 2 : 98 , v / v ), buffer b contained 0 . 1 m teab ( ph 7 . 0 ) in a mixture of acetonitrile and water 60 : 40 ( v / v ) for modified oligonucleotide 8 ( condition i ) and 40 : 60 ( v / v ) for unmodified oligonucleotide 9 ( condition 11 ). flow rate 1 ml / min .= 266 nm . fractions containing the desired product were collected , and the buffer was evaporated under vacuum . the residue was co - evaporated with 96 % ethyl alcohol to remove teab . both oligonucleotides were stored as dry solid at − 20 ° c . when needed , they were redissolved in water , stored as frozen solution , and relyophilized as soon as possible . yield of purified modified oligonucleotides 8 was 9 . 1 a 260 optical density units , and oligonucleotide 9 36 . 9 a 260 optical density units , respectively . 8 : uv ( h20 ): λ min = 233 . 6 nm , λ max = 263 . 1 nu ; rp - hplc ( condition i ) r f = 18 . 65 min ; 9 : uv ( h2o ): λ min = 230 . 0 nm , λ max = 257 . 5 ; rp - hplc ( condition ii ) r f = 12 . 32 min ; maldi - ms 4301 [ m ]. example procedure for the detection of the nucleoside or oligonucleotide labelled with carborane or metallacarborane group . the stock solution of 31 was prepared by dissolving of compound 31 ( 7 , 9 mg , 0 , 02 mmole ) in the mixture of acetonitrile / water ( 40 : 60 v / v , 1 , 5 ml ) containing ammonium acetate ( 0 , 05 m , ph 5 , 5 ), then an aliquot ( 1 μl ) was taken from this solution and was diluted 10 3 - fold with the same as above mixture of solvents up to 1 ml , yielding solution containing 10 ng of compound 31 in 1 , 9 μl of solution . from the diluted as above solution an aliquot ( 1 , 9 μl ) was taken and further diluted 10 - folds up to 20 μl with a mixture acetonitrile / water ( 40 : 60 v / v ) containing ammonium acetate ( 0 , 05 m , ph 5 , 5 ). a final concentration of 31 was 1 , 3 μm corresponding to 0 , 5 ng in 1 μl of solution . this was analyzed by rp - hplc ( econosil rp c 18 , column , 5 μm , 4 , 7 × 250 mm ) using an electrochemical detection in the range of potentials from + 100 mv to + 1400 mv and from − 100 mv to − 600 mv . as eluent a mixture of acetonitrile / water ( 40 : 60 v / v ) containing ammonium acetate ( 0 , 05 m , ph 5 , 5 ) was used . the elution time was 5 min at flow rate 1 ml / min . the analysis was performed at ambient temperature . the stock solution of uridine ( 1 ) was prepared by dissolving 1 ( i mg , 4 μmole ) in a mixture of acetinitrile / water ( 40 : 60 v / v , 1 ml ) containing ammonium acetate ( 0 , 05 m , ph 5 , 5 ). from this solution an aliquot was withdrawn ( 1 μl ) then was diluted 10 3 - fold with the same mixture of solvents up to 1 ml yielding solution containing 10 ng of compounds 1 in 10 μl . from that solution an aliquot was taken ( 10 μl ) then was diluted 2 - fold up to 20 μl by addition of a mixture of acetonitrile / water ( 40 : 60 v / v ) containing ammonium acetate ( 0 , 05 m , ph 5 , 5 ). the final solution containing 2 , 05 μm of nucleoside 1 was analyzed by rp - hplc ( econosil rp c 18 column , 5 μm , 4 , 7 × 250 mm ) using electrochemical detection , the potential range was from + 100 mv to + 1400 mv and from − 100 mv to − 600 mv . as an eluent a mixture of acetonitrile / water ( 40 : 60 v / v ) containing ammonium acetate ( 0 , 05 m , ph 5 , 5 ) was used . elution time was s min , and flow rate was 1 ml / min . the elution was performed at ambient temperature . electrochemical detection of oligonucleotide 5 ′- d ( cgctggittggcu 2 ′- cbm g )- 3 ′ ( 19 ) labelled with 2 ′- o - nido -( o - carboran - 1 - yl ) methyl group . the stock solution of oligonucleotide 19 was prepared by dissolving 0 , 05 odu a260 ( ca . 1 , 65 μg , 0 , 37 nmole ) of the oligomer in a mixture of acetonitrile / water ( 40 : 60 v / v ) ( 0 , 5 ml ) containing ammonium acetate ( 0 , 05 m , ph 5 , 5 ) yielding solution containing 3 × 10 − 4 odu a260 ( ca . 10 ng ) of 19 in 3 μl of solution . from this solution an aliquot ( 3 μl ) was taken and was diluted 7 - fold up to 20 μl by addition of a mixture acetonitrile / water ( 40 : 60 v / v ) containing ammonium acetate ( 0 , 05 m , ph 5 , 5 ). the final solution containing 0 , 12 μm of 19 was analyzed by rp - hplc ( econosil rp c 18 column , 5 μm , 4 , 7 × 250 mm ) using electrochemical detection in the potential ranging from + 100 mv to + 1400 mv and from − 100 mv to − 600 mv . as eluent a mixture of acetonitrile / water ( 40 : 60 v / v ) containing ammonium acetate ( 0 , 05 m , ph 5 , 5 ) was used . the eluting time was 5 min , flow rate was 1 ml / min . the analysis was performed at room temperature . control . an approach to electrochemical detection of unlabeled oligonucleotide 5 ′- d ( cgctggtttggctg )- 3 ′ ( 15 ). the stock solution of 15 was prepared by dissolving 0 , 05 odu a260 ( ca . 1 , 65 μg , 0 , 38 nmole ) of the oligomers 15 in a mixture of acetonitrile / water ( 40 : 60 v / v , 0 , 5 ml ) containing ammonium acetate ( 0 , 05 m , ph 5 , 5 ) yielding solution containing 3 × 10 − 4 odu a260 ( ca . 10 ng , 2 , 3 μmole ) of compound 15 in 3 μl of the solution . from this solution an aliquot ( 3 μl ) was taken and was diluted 7 - fold up to 20 μl with a mixture of acetonitrile / water ( 40 : 60 v / v ) containing ammonium acetate ( 0 , 05 m , ph 5 , 5 ). the final solution 0 , 12 μm of 15 was analyzed by rp - hplc ( econosil rp c 18 column , 5 μm , 4 , 7 × 250 mm ) using electrochemical detection in the potential range from + 100 mv to + 1400 mv and from − 100 mv to − 600 mv . as eluent a mixture of acetonitrile / water ( 40 : 60 v / v ) containing ammonium acetate ( 0 , 05 m , ph 5 , 5 ) was used . the elution time was 5 min , the flow rate was 1 ml / min . the analysis was performed at room temperature . the stock solution of compound 28 was obtained by dissolving 28 ( 0 , 88 mg , 0 , 001 mmole ) in a mixture of acetonitrile / water ( 60 : 40 v / v , 1 ml ) containing ammonium acetate ( 0 , 05 m , ph 5 , 5 ) yielding solution containing 880 ng of 28 in 1 ml of solution . from this solution an aliquot ( 1 μl ) was taken and was diluted 20 - fold up 20 μl with a mixture of acetonitrile / water ( 60 : 40 v / v ) containing ammonium acetate ( 0 , 05 m , ph 5 , 5 ). the final solution containing 67 , 5 μm of 28 was analyzed by rp - hplc system ( fconosil rp c 18 column , 5 μm , 4 , 7 × 250 mm ) equipped with electrochemical detector . the potential range was from + 100 mv to + 2000 mv and from − 100 mv to − 2000 mv . as eluent a mixture of acetonitrile / water ( 60 : 40 v / v ) containing ammonium acetate ( 0 , 05 m , ph 5 , 5 ) was used . the eluting time was 10 min , flow rate 1 ml / min . the analysis was performed at room temperature . the stock solution of thyrnidine was prepared by dissolving thymidine ( 1 mg , 4 , 4 μmole ) in a mixture of acetonitrile / water ( 60 : 40 v / v , 1 ml ) containing ammonium acetate ( 0 , 05 m , ph 5 , 5 ) yielding a solution containing 1 μg of thymidine in 1 μl of solution . from this solution an aliquot was taken ( 1 μl ) and was diluted up to 1 ml with the same mixture of solvents yielding solution containing 10 ng of thymidine in 10 μl of solution , next this solution was further diluted 2 - fold up to 20 μl with same mixture of solvents . the final solution containing 2 , 2 μm of thymidine was analyzed rp - 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