Patent Application: US-50435606-A

Abstract:
this invention relates to the field of nucleic acid chemistry , more specifically to the field of compositions and processes that can bind to nucleic acids , including compositions and processes that by doing so have use in the capture , detection and sequencing of nucleic acids , and most specifically to nucleoside analogs that incorporate nucleobase analogs that are able to bind to different natural nucleobases with nearly equal affinity , and therefore display a degree of ambiguity , or universality , in their binding pattern . such nucleobase analogs , when incorporated into oligonucleotides , allow the oligonucleotides to bind with nearly equal affinity and / or melting temperature , to a complementary strand , template the synthesis of oligonucleotide from primers that have a distribution of sequences , and support the sequencing of oligonucleotides . thus , these have use in human diagnostics , especially when polymorphisms are present in a population .

Description:
two structures , shown in fig1 and fig2 , and named compound i and compound ii , represent pyrimidine - like universal nucleobases that are the objects of the instant invention . a third structure , shown in fig3 and named compound iii , is able to act both like a pyrimidine and like a purine , and is a third object of the instant invention . this invention covers these nucleobases placed on a ribose or ribose analog ( such as 2 ′- o methyl , 2 ′- o allyl , or other modified ribose ring , including deoxyribose ), lna or other ribose analog having the conformational flexibility constraints , pna or other analog of dna with the charges missing , and glycerol dna , glyoxal dna , or other analogs of nucleic acids where an open ring , abbreviated , or other sugar analog is used . the nucleobases are also covered in the instant invention if they are appended to a triphosphate of a sugar , in a form suitable for incorporation into a dna or rna molecule by a polymerase or reverse transcriptase . also covered are oligonucleotides containing the nucleobases in dna analogs that are branched , dendrimeric , cyclic , derivatized . ( e . g ., with a fluorescent tag ) and / or immobilized . the strategy disclosed in this specification for inventing a pyrimidine - like universal nucleobase was to ensure that the nucleobase analog can present a full hydrogen bonding pattern to a standard purine on the complementary strand , where two tautomeric forms present the two different hydrogen bonding patterns . in the design of the molecule , the goal is to have the two tautomeric forms have as nearly equal energies as possible , so that the equilibrium constant between the two is as near to unity as possible . this requires the construction of molecules that have high degrees of symmetry . while perfect symmetry is not possible , as there is only one ribose ring , if the ribose is assumed to contribute to electronic distribution as a standard alkyl group , an alkyl group at the appropriately symmetric position create the appropriate symmetry . the design of pyrimidine - like universal nucleobases also takes advantage of the extensive studies that show that most dna polymerases are largely insensitive to the placement of bulky side chains in the major groove . this has been exploited for over two decades in dna sequencing and dna labeling . the ability of ligases to accept large substituents in the major group is less well studied , but this region also appears to tolerate substitution at nucleotides at positions other than the nucleotide that is directly participating in the ligation reaction . several features of these compounds should be noted . first , compound one is a c - glycoside . this means that the heterocyclic nucleobase is joined to the sugar by a carbon - carbon bonds , rather than the carbon - nitrogen bond that joins the nucleobase to the sugar in a standard nucleotide . preliminary work at the foundation has shown that some polymerases , such as therminator , will accept a string of 12 c - glycosides in row . obviously , an applications here , the level of c - glycoside incorporation will be lower . compound ii contains a positive charge in the nucleobase period through resonance , this positive charge is distributed up and down the two fused ring systems . geyer et al . [ gey03 ] showed that introduction of a positive charge into a nucleobase stabilized the duplex modesty . we do not expect , of course , that multiple stacked cationic nucleobases would be desirable . the corresponding species without the methyl group on the nitrogen does not have a positive charge . it lacks , however , some of the symmetry that ensures that the tautomeric equilibrium constant is close to unity . fig1 and 2 shows the rationales by which compounds i and compounds ii were designed . both structures display high levels of symmetry and electronic distribution that is necessary to ensure that two tautomeric forms are close in energy . inspection of the structure shows that in each case , one of the tautomeric forms presents the hydrogen bond acceptor - donor - acceptor pattern characteristic of thymidine , while the other presents the hydrogen bonding pattern donor - acceptor - acceptor characteristic of cytidine . because of the symmetry , it is likely that a dna incorporating compound one or compound to at a site will bind with nearly equal affinity to adenosine and guanosine in the complementary nucleotide at the paired site . as the modification involves only the introduction of extra material into the major groove , we expect compound i and compound ii in a template to be accepted by most polymerases . we also expect that these compounds will be accepted by ligase in the five paired nucleotide that bind in the active site . it remains an open question , to be determined in phase 1 work , whether either compound can be excepted by a ligase at the site of ligation . it is interesting to note that two decades ago , inoue et al . prepared a pyridopyrimidine structure that is similar to the structure of the compound ii [ ino85 ] ( specifically , 3 - beta - d - 2 ′- deoxyribofuranosyl - 2 , 7 - dioxopyrido [ 2 , 3 - d ] pyrimidine and its ribo counterpart ). this compound lack the second nitrogen , and therefore the full symmetry of compound ii . curiously , although these authors report that the compound forms hydrogen bonds with g ( more strongly ) and a ( more weakly ), they evidently did not recognize the tautomeric ambiguity of the species , nor the asymmetry in the compound , nor that the differences in binding affinity might arise because adenine presents only two hydrogen bonding opportunities , while guanine presents three . instead , they propose that the binding to a is via a wobble structure . the same analysis is involved in analysis of iii , with the additional observation that it can exist in either the syn conformer around the glycosidic bond , or the anti conformer . in the syn conformer , tautomeric forms exist that are complementary , in a hydrogen bonding sense , to a and g . in the anti conformer , tautomeric forms exist that are complementary , in a hydrogen bonding sense , to t and c . further , we disclose that the sugar that carries the nucleobase analog heterocycle can be ( preferably ) 2 ′- deoxyribose . more stable binding can be had , however , if the sugar is ribose , or one of its hydrolytically stable variants , including 2 ′ o - methyl ribose . in addition , we disclose that the glycerol sugar analogs of zhang et al . [ zha05 ] are suitable to support these sugars . further , because the pyrimidine analogs lack hydrogen bonding opportunities in the major groove , they can be supported on a pna backbone . this is not the case , however , for structure iii . the starting procedure for the compound represented by structure ii is 5 - formyl - 2 ′- deoxycytidine ( fig6 ). this is prepared using the procedure presented by abdel rahman in 2001 [ abd01 ]. other sources of this compound are found in [[ bie96 ] [ ita91 ] [ ita94 ]] [ laf98 ] [ laf00 ] [ mur99 ], and it is also known as a natural product [ kas98 ][ kaw94 ][ mor94 ], [ tom97 ] and is known as the 2 ′- ome riboside as well [ pai96 ] [ ] [ ]. commercially available 5 - methyl - 2 ′- deoxycytidine ( 30 mmol ) is dissolved in dry pyridine ( 100 ml ), and the solution is cooled on ice . to this is added tbutyldimethylsilyl chloride ( 33 mmol ). after incubation at 0 ° c . for 5 hours , the reaction is quenched by the evaporation of the solvents under high vacuum at temperatures less than 40 ° c . the residue is then suspended in a mixture of water and ether , the ether layer is recovered , and the layer is dried and evaporated to give the 5 ′ pmethyl - 2 ′- deoxycytidine as its 5 ′- tbutyldimethylsilyl ether ; the product from the step above was treated with na 2 s 2 o 8 to give a mixture of 5 -( hydroxymethyl )- and 5 - formyl - 2 ′- deoxycytidine as products . these are separated by chromatography ( silica , etoac ). the second product is the precursor of the the hydroxymethyl group of the first product ( 4 mmol ) is oxidized to a formyl group by treatment with ceric ( iv ) ammonium nitrate ( can , 1 m ) at 60 ° c . for 1 h . this product is also purified by chromatography ( silica , etoac ). the 5 ′- formyl - 2 ′- deoxycytidine ( 5 mmol ), after coevaporation with dry pyridine , is converted to the 3 ′- 5 ′- bis ( trimethylsilyl ether by reaction with bistrimethylsilyl acetamide ( 2 . 2 equiv ) at 0 ° c . in dry pyridine . the reaction mixture is then directly treated with methyl isocyanate , ( 1 . 05 equiv , 0 ° c ., 2 hours ) to yield ii . the product is purified by column chromatography ( silica , etoac ). this nucleoside analog is used as the starting point for the preparation of an activated phosphoramidite for oligonucleotides analog synthesis . the heterocycle itself need not be protected , as it does not carry any exocyclic amino groups , but the 5 ′- oh group must be protected , as always . thus , the nucleoside analog ( 17 . 4 mmol ) is dissolved in dry pyridine ( 250 ml ). to the solution is added 4 ′, 4 ″- dimethoxytrityl chloride ( 1 . 2 equiv ). the reaction mixture is stirred at room temperature for 24 hours . the reaction is then quenched by the addition of water ( 6 ml ). the solution is concentrated under vacuum , and an aqueous solution of nahco 3 ( 150 ml ) is added . the mixture is extracted with etoac , dried ( na 2 so 4 ), the solvents evaporated under reduced pressure , and the product isolated by column chromatography ( chloroform / acetone 9 : 1 , then 9 : 2 ). the product from above ( 0 . 12 mmol ) is dissolved in ch 3 cn ( 2 . 0 ml ). the solution is then treated with bis -( n , n - diisopropylamino )- 3 - cyanoethyloxyphosphine ( aldrich , 1 . 2 equiv . ), and diisopropylammonium tetrazolide ( 0 . 06 mmol ), following a literature procedure [ mcbride , l . j ., kierzek , r ., beaucage , s . l . & amp ; caruthers , m . h . ( 1986 ) j . am . chem . soc . 108 , 2040 - 2048 ]. the progress of the reaction is monitored by tlc ( sio 2 eluted with etoac : ch 2 cl 2 : triethylamine 45 : 45 : 10 ). an additional portion ( 0 . 02 ml ) of bis -( n , n - diisopropylamino )- 3 - cyanoethyloxyphosphine is then added , and stirring continued for an additional hour . water ( 2 drops ) is added , the mixture stirred for 15 min , the mixture diluted with ch 2 cl 2 ( 30 ml ), and the organic layer washed with aqueous na 2 co 3 ( 2 %) and dried ( na 2 so 4 ). the phosphoramidite ( 120 . 3 mg , 93 %) is isolated by chromatography ( sio 2 , etoac : ch 2 cl 2 : triethylamine 45 : 45 : 10 as eluant ). alternatively , the protected species is be converted to its triphosphate via the procedure of ludwig and eckstein [ lud89 ]. in this procedure , the first intermediate is the 3 ′- protected acetate . the product is then detritylated to yield the 5 ′- deprotected - 3 ′- acetate as the intermediate that is then used to generate a triphosphate . the heterocycle found in compound i is known from the work of stoss et al . [ stoss91 ]. it is also was prepared by the condensation of guanidine and 3 - methoxyacrylate esters [ tan02 ]. synthesis of i from that heterocycle involves coupling of the iodinated heterocycle to the known glycal prepared from thymidine ( fig7 ). an alternative approach is available to prepare the nucleoside carrying this heterocycle ( fig8 ). here , the commercially available 2 - amino - 4 ( 1h )- pyrimidinone ( isocytidine ) is phenoxyacetylated and treated with the t - butyl ester of propargylic acid . this gives a product with the double bond having primarily the trans configuration . the phenoxyacetyl group is removed to generate the free amine . this closes spontaneously when the double bond is in the cis form , upon michael addition by thioanisol to give the beta thioether ( fig8 ). the heterocycle ( 14 mmol ) is rendered anhydrous by coevaporation with dmf under high vacuum . the dry material is then dissolved in dmf ( 50 ml ). n - iodosuccinimide ( 4 . 73 g ; 21 mmol ) is added and the resulting solution is stirred at room temperature in the dark . after 4 days , the solution is slowly added to vigorously stirred water ( 2 l ) and the resulting suspension is stirred overnight . the solid iodinated heterocycle is recovered by filtration and dried in a vacuum over p 2 o 5 gives the product as a yellow solid . the iodinated heterocycle is then coupled to 3 - tertbutyldiphenylsilyloxy - 2 - hydroxymethyl - 2 , 3 - dihydrofuran , which is well known in the literature [ hut03 ] [ ire80 ] [ lar94 ] [ wal96 ] [ cam97 ], and referred to here as simply the “ glycal ”. the heck coupling is done following literature procedures , using palladium acetate with triphenylarsine as the catalyst system , and anhydrous dimethylformamide ( dmf ) as the solvent . the coupling to the glycal and subsequent deprotection and reduction have been reported previously by several groups [ farr , r . n . outten , r . a . cheng , j . c .- y . daves , jr ., g . d . ( 1990 ) organometallics 9 , 3151 ][ zhang , h .- c . daves , jr ., g . d . ( 1992 ) j . org . chem . 57 , 4690 ][ zhang , h .- c . daves , jr ., g . d . ( 1993 ) organometallics 12 , 1499 ][ hsieh , h .- p . mclaughlin , l . w . ( 1995 ) j . org . chem ., 60 , 5356 ][ chen , d . l . mclaughlin , l . w . ( 2000 ) j . org . chem ., 65 , 7468 . ][ searls , t . chen , d . l . lan , t . mclaughlin , l . w . ( 2000 ) biochemistry , 39 , 4375 . ][ lan , t . ; mclaughlin , l . w . ( 2001 ) bioorg . chem . 29 , 198 ] [ coleman , r . s ., madaras , m . l . ( 1998 ) j . org . chem . 63 , 5700 ]. triethylamine is often used instead of tributylamine as the base , since it is easier to remove during purification . thus , a suspension of palladium acetate ( 7 mg , 0 . 031 mmol ) and triphenyl arsine ( 19 mg , 0 . 061 mmol ) in dmf ( 2 ml ) is stirred at room temperature for 30 min , to give a yellow suspension . a solution of the iodinated heterocycle ( 0 . 204 mmol ) and the 3 - tbdps - 5 - unprotected glycal ( 109 mg , 0 . 307 mmol ) is prepared in dmf ( 3 ml ) and dried overnight over molecular sieves ( 4 å ). this solution is added to the yellow suspension , followed immediately by addition of triethylamine ( 0 . 06 ml , 0 . 408 mmol ). the suspension is stirred at 55 ° c . for 2 days . the solvent is removed under high vacuum . the residue is suspended in ch 2 cl 2 ( 60 ml ), the insoluble material removed by filtration and the organic solution evaporated . column chromatography on silica ( hexane : etoac = 2 : 1 to 1 : 1 . 5 as eluant gives the coupled product , with some of the tbdps removed . therefore , the mixture is reduced rapidly ( net step ). sodium triacetoxyborohydride ( 0 . 549 g , 2 . 475 mmol ) is added in one lot to a solution of the product of the previous step ( 1 . 65 mmol ) in acetonitrile ( 8 ml ) and acetic acid ( 4 ml ) under an ar atmosphere . tlc ( ethyl acetate ) indicated that there is no starting material after 12 min . the reaction is quenched by the addition of acetone , and the reaction mixture concentrated under reduced pressure . the resulting pale yellow gum is dissolved in methanol , adsorbed onto silica , and purified by flash chromatography ( ethyl acetate ) to give the desired material as a white solid . this nucleoside analog is used as the starting point for the preparation of an activated phosphoramidite for oligonucleotides analog synthesis . the heterocycle itself need not be protected , but the 5 ′- oh group must be protected , as always . thus , the nucleoside analog ( 8 . 7 mmol ) is dissolved in dry pyridine ( 150 ml ). to the solution is added 4 ′, 4 ″- dimethoxytrityl chloride ( 1 . 2 equiv ). the reaction mixture is stirred at room temperature for 24 hours . the reaction is then quenched by the addition of water ( 3 ml ). the solution is concentrated under vacuum , and an aqueous solution of nahco 3 ( 80 ml ) is added . the mixture is extracted with etoac , dried ( na 2 so 4 ), the solvents evaporated under reduced pressure , and the product isolated by column chromatography ( chloroform / acetone 9 : 1 , then 9 : 2 ). the product from above ( 0 . 12 mmol ) is dissolved in ch 3 cn ( 2 . 0 ml ). the solution is then treated with bis -( n , n - diisopropylamino )- 3 - cyanoethyloxyphosphine ( aldrich , 1 . 2 equiv . ), and diisopropylammonium tetrazolide ( 0 . 06 mmol ), following a literature procedure [ mcbride , l . j ., kierzek , r ., beaucage , s . l . & amp ; caruthers , m . h . ( 1986 ) j . am . chem . soc . 108 , 2040 - 2048 ]. the progress of the reaction is monitored by tlc ( sio 2 eluted with etoac : ch 2 cl 2 : triethylamine 45 : 45 : 10 ). an additional portion ( 0 . 02 ml ) of bis -( n , n - diisopropylamino )- 3 - cyanoethyloxyphosphine is then added , and stirring continued for an additional hour . water ( 2 drops ) is added , the mixture stirred for 15 min , the mixture diluted with ch 2 cl 2 ( 30 ml ), and the organic layer washed with aqueous na 2 co 3 ( 2 %) and dried ( na 2 so 4 ). the phosphoramidite ( 120 . 3 mg , 93 %) is isolated by chromatography ( sio 2 , etoac : ch 2 cl 2 : triethylamine 45 : 45 : 10 as eluant ). alternatively , the protected species is be converted to its triphosphate via the procedure of ludwig and eckstein [ lud89 ]. in this procedure , the first intermediate is the 3 ′- protected acetate . the product is then detritylated to yield the 5 ′- deprotected - 3 ′- acetate as the intermediate that is then used to generate a triphosphate . a compound that serves as both a universal purine and pyrimidine nucleobase a route for the synthesis of iii is shown in fig9 from a structure that is reported in the literature [ graboyes et al . ( 1968 ) j . med . chem . 11 , 568 - 573 ]. this compound ( 2 . 8 mmols ) is covaporated with pyridine three times to ensure that it is dry . the compound is then dissolved in dry pyridine ( 10 ml ), and the solution is treated with ch 3 nco ( 2 . 1 equiv ., r = me ). this material is then suspended in phosphorous oxychloride ( 10 ml , 0 ° c . ), and the mixture is allowed to warm to room temperature , and then under ar to 80 ° c . to give the heterocycle . this heterocycle is then converted to a riboside , a riboside derivative , or a 2 ′- deoxyriboside using any standard electrophile . for the case in fig9 , a slurry of the heterocycle ( 17 . 8 mmol ), k 2 co 3 ( 58 mmol ), and tris [ 2 -( 2 - methoxyethoxy ) ethyl ] amine ( 0 . 20 ml , 0 . 42 mmol ) in anhydrous acetonitrile ( 1000 ml ) is stirred at room temperature for 1 h . at this time , bis - tolyl protected 1 - chloro - 2 ′- deoxyribose ( berry & amp ; associates , 9 g , 23 . 2 mmol ) is added . the stirring is continued for two hours . after the coupling is complete , the solids are removed by filtration , the filtrate is recovered , and the solvents are removed under vacuum . the product present in the residue is purified by column chromatography on silica gel using 1 - 2 % me 2 co / ch 2 cl 2 as eluant to yield the title compound as a white foam , along with a mixture of other isomers . ireland , r . e ., thaisrivongs , s ., vanier , n ., wilcox , c . s . 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