Patent Application: US-201213439179-A

Abstract:
this invention provides compounds that are of the class of nucleoside analogs , and more specifically nucleoside analogs that have a non - standard nucleobase , and more specifically analogs where a side chain is appended to said non - standard nucleobase , where said side chain can be cleaved so as to leave behind no more than five atoms appended to the nucleobase as a “ scar ”. the claimed compounds are useful as intermediates in processes that transiently introduce tags , labels , fluorescent molecules , or other species into oligonucleotides , as in sequencing using cyclic reversible termination , in in vitro selection using functionalized nucleotides that must later be pcr amplified , or in nucleotide capture protocols used in diagnostics .

Description:
two architectures are disclosed here for creating nucleotides that , once incorporated into an oligonucleotide , can be cleaved without leaving a scar . the first architecture , illustrated in fig1 , involves a “ push ” from the nucleobase to force a leaving group in the linker to depart , where the ability of the linker to depart is enhanced by added reagents that enhance the ability of a fragment of the linker to act as a leaving group . following its departure , water from solvent adds back to the product to create a hydroxymethyl group replacing the linker . this is implemented with either standard pyrimidines or 9 - deazapurines . the second architecture involves a “ push ” from the linker after it is transformed by a chemical reaction . illustrated in fig2 with thioether linkages and , in its presently preferred form , in fig3 with a 1 , 2 - diol in the linker , this second class has the nucleobase itself be a leaving group . while not wishing to be bound by theory , the inventive step is to recognize that a c - glycoside can allow the linkage to be attached to a nitrogen whose conjugate acid form has a pka in a range from approximately 8 to approximately 10 . the pka &# 39 ; s of several of the nucleobase analogs that implement this invention is found in the literature . for example , luyten et al . summarized the literature ( see references therein ) and reported new pka &# 39 ; s for formycin a ( 9 . 6 , for deprotonation of n7 ), formycin b ( 10 . 4 for the deprotonation of n7 ), pseudoisocytidine ( 9 . 0 for the deprotonation of n3 ) and pseudouridine ( 9 . 1 , cite of deprotonation unclear ) [ luyten et al , 1997 ]. adding another nitrogen to a ring ( e . g ., proceeding from 9 - deazapurines to 8 - aza - 9 - deazapurines or from pseudouridine to 6 - azapseudouridine or , correctly numbered , 4 - azapseudouridine ) lowers the relevant pka by 1 - 2 pka units . showing that this inventive concept was reducible to practice , 1 , 2 - diol linkers were prepared for pseudouracil , an analog of the nucleobase in natural thymidine . cleavage of the 1 , 2 - diol with periodate generated an aldehyde , which readily fragmented at modest ph to give pseudouracil derivatives with a half life of just minutes . the structure of the linker past the cleavable functionality is incidental to the invention . thus , that linker can carry any kind of tag , including a fluorescent tag , a redox active tag , and a mass spectral tag . further , the linker can be an aminoalkyl group ( as in example 1 ), a carboxyalkyl group , a thioalkyl group , or a hydroxyalkyl group . this disclosure teaches that an additional nitrogen can be incorporated at either position 6 in the pyrimidines ( using analogous numbering to the standard nucleobases ) or at position 8 in the purines ( using analogous numbering to the standard nucleobases ). this additional nitrogen has the effect of making the beta elimination process faster , at the cost of making the product more “ unnatural ”. the nucleoside analogs with the tags and the nucleobases appropriately protected and the 2 ′- oh group acetylated , as in the standard ludwig - eckstein procedure , can be converted into triphosphates using that procedure [ ludwig et al , 1989 ]. an additional unexpected discovery was then made when testing polymerases with triphosphates of c - glycosides , 2 ′- deoxypseudouridine in particular , with side chains that would generate an aldehyde unit that could fragment , as shown in fig7 , to give a “ thymine ”. here , polymerases were found that did not need a 3 ′- onh 2 unit to stop extension after a single nucleotide incorporation . this is analogous to the metzker approach mentioned in the introduction of using a base modification , rather than a 3 ′- block to achieve reversible termination . however , unlike the metzker modifications , the modifications disclosed here are attached to what would be formally the 5 - position ( by analogy to the numbering system used in standard nucleotides ). further , they are removable by a chemical step , not a photochemical step . to a stirred suspension of 2 - deoxy - d - ribose ( 5 g , 37 . 3 mmol ) in thf ( 100 ml ) were added p - toluenesulfonic acid ( 150 mg , 0 . 79 mmol ) and 2 , 2 - dimethoxypropane ( 10 ml ) at room temperature . the mixture was stirred at room temperature for 45 min , neutralized with et 3 n and concentrated . the residue was diluted with etoac and extracted with h 2 o . the organic layer was dried ( na 2 so 4 ), filtered and concentrated . the residue was resolved by silica gel column chromatography ( hex / etoac = 1 / 2 ) to give compound 1 ( 4 . 26 g , 24 . 5 mmol , 66 %) as an oil . 1 h nmr ( cdcl 3 , 300 mhz ) δ 5 . 21 ( m , 1h ), 4 . 44 ( m , 1h ), 4 . 13 ( m , 1h ), 3 . 91 ( m , 1h ), 3 . 65 ( m , 1h ), 3 / 23 ( m , 1h ), 2 . 19 - 2 . 27 ( m , 1h ), 1 . 72 - 1 . 91 ( m , 1h ), 1 . 49 , 1 . 33 ( 2s , 6h ). to a stirred suspension of methyltriphenylphosphonium bromide ( 24 . 86 g , 69 . 6 mmol ) in thf ( 180 ml ) was added 2 . 5m solution of buli in hexane ( 27 . 8 ml , 69 . 5 mmol ) at this mixture was stirred at room temperature for 1 h and cooled to 0 ° c . a solution of compound 1 ( 4 . 04 g , 23 . 2 mmol ) in thf ( 30 ml ) was added to this mixture at 0 ° c . the reaction mixture was stirred at room temperature for 2 h and aq . ammonium chloride solution ( 100 ml ) was added . the mixture was extracted with etoac . the organic layer was washed with brine , dried ( na 2 so 4 ), filtered and concentrated . the residue was resolved by silica gel column chromatography ( hex / etoac = 1 / 1 ) to give compound 2 ( 3 . 1 g , 18 mmol , 78 %) as an oil . 1 h nmr ( cdcl 3 , 300 mhz ) δ 5 . 76 - 5 . 89 ( m , 1h ), 5 . 07 - 5 . 17 ( m , 2h ), 4 / 14 - 4 / 28 ( m , 2h ), 3 . 61 - 3 . 65 ( m , 2h ), 2 . 22 - 2 . 44 ( m , 2h ), 2 . 05 ( t , 1h , j = 6 . 0 ), 1 . 47 , 1 . 36 ( 2s , 6h ); 13 c nmr ( cdcl 3 , 75 mhz ) δ 134 . 4 , 116 . 6 , 108 . 5 , 78 . 0 , 76 . 5 , 61 . 8 , 33 . 9 , 28 . 4 , 25 . 7 . to a stirred solution of compound 2 ( 2 . 8 g , 16 . 3 mmol ) in thf ( 120 ml ) were added pph 3 ( 6 . 4 g , 24 . 4 mmol ) and dead ( 40 % in toluene , 10 . 6 ml , 24 . 3 mmol ) at 0 ° c . after 20 min stirring , phthalimide ( 3 . 59 g , 24 . 4 mmol ). the reaction mixture was stirred overnight at room temperature , concentrated , diluted with etoac ( 300 ml ) and extracted with aq . nahco 3 solution ( 100 ml ). the organic layer was dried ( na 2 so 4 ), filtered and concentrated . the residue was purified by silica gel column chromatography ( hex / etoac = 2 / 1 ) to give compound 3 ( 4 . 74 g , 15 . 7 mmol , 97 %) as an oil . 1 h nmr ( cdcl 3 , 300 mhz ) δ 7 . 80 - 7 . 85 ( m , 2h ), 7 . 68 - 7 . 73 ( m , 2h ), 5 . 82 ( m , 1h ), 5 . 13 - 5 . 24 ( m , 2h ), 4 . 48 ( m , 1h ), 4 . 26 ( m , 1h ), 3 . 90 ( dd , 1h , j = 10 . 5 , 13 . 5 ), 3 . 52 ( dd , 1h , j = 3 . 0 , 13 . 5 ), 2 . 37 - 2 . 57 ( m , 2h ), 1 . 53 , 1 . 30 ( 2s , 6h ); 13 c nmr ( cdcl 3 , 75 mhz ) δ 168 . 5 , 134 . 1 , 133 . 9 , 132 . 4 , 123 . 5 , 118 . 0 , 109 . 1 , 76 . 5 , 74 . 3 , 39 . 1 , 33 . 5 , 28 . 2 , 26 . 1 . to a stirred solution of compound 3 ( 4 . 74 g , 15 . 7 mmol ) in etoh ( 120 ml ) was added hydrazine hydrate ( 3 ml ) at room temperature . the reaction mixture was stirred at 60 ° c . for 3 h , cooled to rt and filtered ( washed with ch 2 cl 2 ). the filtrate was concentrated to give crude compound 4 , which was dissolved in ch 2 cl 2 ( 80 ml ). to this solution were added et 3 n ( 4 . 38 ml , 31 . 4 mmol ) and ( boc ) 2 o ( 3 . 44 g , 15 . 8 mmol ) at room temperature . the reaction mixture was stirred at room temperature for 3 h , poured into sat . nahco 3 sol &# 39 ; n and extracted with ch 2 cl 2 . the organic layer was dried ( na 2 so 4 ), filtered and concentrated . the residue was resolved by silica gel column chromatography ( hex / etoac = 4 / 1 to 3 / 1 ) to give compound 5 ( 2 . 75 g , 10 . 1 mmol , 64 %) as an oil . 1 h nmr ( cdcl 3 , 300 mhz ) δ 5 . 75 ( m , 1h ), 5 . 08 - 5 . 18 ( m , 2h ), 4 . 85 ( m , 1h ), 4 . 17 ( m , 1h ), 4 . 09 ( m , 1h ), 3 . 41 ( m , 1h ), 2 . 93 ( m , 1h ), 2 . 20 - 2 . 40 ( m , 2h ), 1 . 45 ( s , 3h ), 1 . 43 ( s , 9h ), 1 . 33 ( s , 3h ); 13 c nmr ( cdcl 3 , 75 mhz ) δ 156 . 1 , 134 . 1 , 117 . 8 , 108 . 5 , 79 . 7 , 76 . 6 , 41 . 1 , 33 . 8 , 28 . 6 , 28 . 5 , 25 . 9 . compound 5 ( 2 . 75 g , 10 . 1 mmol ) was dissolved in 1 / 1 / 1 mixture of thf , acetone and aq . ph 7 . 0 buffer ( 45 ml ) at room temperature . nmo ( 1 . 56 g , 13 . 3 mmol ) was added followed by oso 4 ( 2 . 5 % in t - butanol , 12 . 5 ml ) at rt . the reaction mixture was stirred overnight at room temperature , and sodium bisulfite ( 10 . 13 g ) in water ( 150 ml ) was added . the mixture was stirred for 10 min and extracted with etoac (× 2 ). the combined organic layers were dried ( na 2 so 4 ), filtered and concentrated to give crude compound 6 . compound 6 was dissolved in thf ( 80 ml ) and a solution of naio 4 ( 4 . 28 g , 20 mmol ) in aq . ph 7 . 0 buffer ( 60 ml ) was added at rt . after 1 . 5 h stirring , nabh 4 ( 1 g , 26 . 4 mmol ) at room temperature . the reaction mixture was stirred at room temperature for 1 h and partitioned between etoac and brine . the organic layer was dried ( na 2 so 4 ), filtered and concentrated . the residue was resolved by silica gel column chromatography ( hex / etoac = 3 / 7 ) to give compound 7 ( 2 . 29 g , 8 . 38 mmol , 83 %) as an oil . 1 h nmr ( cdcl 3 , 300 mhz ) δ 4 . 90 ( m , 1h ), 4 . 28 ( m , 1h ), 4 . 11 ( m , 1h ), 3 . 73 - 3 . 84 ( m , 2h ), 3 . 38 ( m , 1h ), 2 . 92 ( m , 1h ), 2 . 42 ( dd , 1h , j = 4 . 2 , 6 . 6 ), 1 . 63 - 1 . 80 ( m , 2h ), 1 . 44 ( s , 3h ), 1 . 42 ( s , 9h ), 1 . 33 ( s , 3h ); 13 c nmr ( cdcl 3 , 75 mhz ) δ 156 . 2 , 108 . 7 , 79 . 8 , 77 . 0 , 76 . 1 , 61 . 1 , 41 . 3 , 31 . 7 , 28 . 6 , 28 . 5 , 25 . 9 . to a stirred solution of compound 7 ( 1 . 77 g , 6 . 5 mmol ) in thf ( 60 ml ) was added n 3 - bz - 5 - iodouracil ( 2 . 43 g , 7 . 1 mmol ). after 10 min , pph 3 ( 1 . 86 g , 7 . 1 mmol ) was added . after 10 min , dead ( 40 % in toluene , 3 . 1 ml , 7 . 1 mmol ) was added . the mixture was stirred at room temperature for 3 h and concentrated . the residue was resolved by silica gel column chromatography ( hex / etoac = 1 / 1 ) to give compound 8 ( 2 . 7 g , 4 . 5 mmol , 70 %) as a foam . 1 h nmr ( cdcl 3 , 300 mhz ) δ 7 . 88 - 7 . 91 ( m , 2h ), 7 . 78 ( s , 1h ), 7 . 63 ( m , 1h ), 7 . 47 ( t , 2h , j = 7 . 8 ), 4 . 82 ( m , 1h ), 4 . 00 - 4 . 16 ( m , 3h ), 3 . 79 ( m , 1h ), 3 . 35 ( m , 1h ), 2 . 93 ( m , 1h ), 1 . 82 - 1 . 95 ( m , 2h ), 1 . 46 ( s , 3h ), 1 . 42 ( s , 9h ), 1 . 34 ( s , 3h ); 13 c nmr ( cdcl 3 , 75 mhz ) δ 168 . 0 , 159 . 6 , 156 . 0 , 149 . 7 , 149 . 5 , 135 . 5 , 131 . 2 , 130 . 8 , 129 . 5 , 109 . 1 , 79 . 9 , 76 . 7 , 74 . 1 , 67 . 1 , 47 . 8 , 41 . 1 , 28 . 6 , 28 . 5 , 28 . 3 , 25 . 8 . a solution of compound 8 ( 1 . 37 g , 2 . 29 mmol ) in 7n nh 3 in meoh ( 40 ml ) was stirred overnight at room temperature and concentrated . the residue was resolved by silica gel column chromatography ( hex / etoac = 1 / 2 ) to give compound 9 ( 800 mg , 1 . 62 mmol , 71 %) as a white foam . 1 h nmr ( cdcl 3 , 300 mhz ) δ 9 . 22 ( brs , 1h ), 7 . 69 ( s , 1h ), 4 . 88 ( m , 1h ), 3 . 99 - 4 . 18 ( m , 3h ), 3 . 73 ( m , 1h ), 3 . 38 ( m , 1h ), 2 . 93 ( m , 1h ), 1 . 91 ( m , 1h ), 1 . 74 ( m , 1h ), 1 . 44 ( s , 3h ), 1 . 43 ( s , 9h ), 1 . 334 ( s , 3h ); 13 c nmr ( cdcl 3 , 75 mhz ) δ 160 . 8 , 156 . 1 , 150 . 6 , 149 . 8 , 109 . 0 , 80 . 0 , 76 . 7 , 73 . 8 , 67 . 5 , 47 . 3 , 41 . 2 , 28 . 6 , 28 . 1 , 25 . 8 . a mixture of pd ( oac ) 2 ( 190 mg , 0 . 85 mmol ) and asph 3 ( 519 mg , 1 . 69 mmol ) in dmf ( 45 ml ) was stirred at room temperature for 30 min . to this mixture was added a solution of glycal 10 ( 2 . 4 g , 6 . 77 mmol ), compound 9 ( 2 . 8 g , 5 . 65 mmol ) and bu 3 n ( 2 . 67 ml , 11 . 3 mmol ) in dmf ( 30 ml ). the reaction mixture was stirred at 60 ° c . for 18 h , cooled to room temperature and concentrated . the residue was resolved by silica gel column chromatography ( hex / etoac = 3 / 7 ) to give compound 11 ( 1 . 9 g , 2 . 64 mmol , 47 %) as a yellow foam . 1 h nmr ( cdcl 3 , 300 mhz ) δ 7 . 80 ( m , 2h ), 7 . 71 ( m , 2h ), 7 . 39 - 7 . 47 ( m , 7h ), 7 / 09 ( brs , 1h ), 5 . 53 ( m , 1h ), 4 . 91 ( m , 1h ), 4 . 69 ( m , 1h ), 4 . 08 - 4 . 20 ( m , 3h ), 3 . 80 - 3 . 93 ( m , 2h ), 3 . 73 ( m , 2h ), 2 . 98 - 3 . 42 ( m , 3h ), 1 . 79 ( m , 2h ), 1 . 42 ( s , 9h ), 1 . 40 ( s , 3h ), 1 . 28 ( s , 3h ), 1 . 05 ( s , 9h ). to a stirred solution of compound 11 ( 1 . 9 g , 2 . 64 mmol ) in thf ( 25 ml ) were added acoh ( 0 . 75 ml ) and 1m tbaf in thf ( 3 . 95 ml , 3 . 95 mmol ) at 0 ° c . after being stirred at 0 ° c . for 1 h , the reaction mixture was concentrated to give crude compound 12 . compound 12 was dissolved in ch 3 cn / acoh ( 2 / 1 , 30 ml ) and na ( oac ) 3 bh ( 838 mg , 3 . 95 mmol ) was added at 0 ° c . the reaction mixture was stirred at 0 ° c . for 1 h and acetone ( 5 ml ) was added . after the solvent was removed , the residue was resolved by silica gel column chromatography ( ch 2 cl 2 / meoh = 10 / 1 to 7 / 1 ) to give compound 13 ( 700 mg , 1 . 44 mmol , 55 %) as a white solid . 1 h nmr ( dmso - d 6 , 300 mhz ) δ 11 . 29 ( brs , 1h ), 7 . 55 ( s , 1h ), 6 . 82 ( t , 1h , j = 5 . 7 ), 4 . 96 ( d , 1h , j = 3 . 6 ), 4 . 75 ( m , 1h ), 4 . 69 ( t , 1h , j = 5 . 7 ), 3 . 99 - 4 . 09 ( m , 3h ), 3 . 80 ( m , 1h ), 3 . 60 - 3 . 69 ( m , 2h ), 3 . 39 ( d , 2h , j = 5 . 1 ), 3 . 14 ( d , 1h , j = 3 . 6 ), 3 . 02 ( m , 1h ), 2 . 89 ( m , 1h ), 1 . 96 - 2 . 02 ( m , 1h ), 1 . 34 ( s , 9h ), 1 . 32 ( s , 3h ), 1 . 21 ( s , 3h ); 13 c nmr ( dmso - d 6 , 75 mhz ) δ 163 . 6 , 158 . 2 , 151 . 2 , 142 . 4 , 114 . 2 , 108 . 1 , 87 . 8 , 78 . 5 , 76 . 1 , 74 . 8 , 73 . 9 , 72 . 8 , 62 . 9 , 41 . 4 , 41 . 0 , 39 . 4 , 28 . 9 , 26 . 4 . to a stirred solution of compound 13 ( 690 mg , 1 . 42 mmol ) in dmf ( 15 ml ) were added imidazole ( 291 mg , 4 . 28 mmol ) and tbdpsc1 ( 0 . 45 ml , 1 . 72 mmol ) at 0 ° c . the reaction mixture was attired at room temperature overnight and concentrated . the residue was resolved by silica gel column chromatography ( hex / etoac = 1 / 4 ) to give compound 14 ( 780 mg , 1 . 08 mmol , 76 %) as a white foam . 1 h nmr ( cdcl 3 , 300 mhz ) δ 9 . 17 ( brs , 1h ), 7 . 64 - 7 . 69 ( m , 4h ), 7 . 35 - 7 . 43 ( m , 6h ), 7 . 29 ( s , 1h ), 5 . 02 ( dd , 1h , j = 9 . 4 , 9 . 9 ), 4 . 85 ( m , 1h ), 4 . 49 ( m , 1h ), 3 . 99 - 4 . 10 ( m , 31 - 1 ), 3 . 66 - 3 . 84 ( m , 4h ), 3 . 34 ( m , 1h ), 2 . 90 ( m , 1h ), 2 . 43 ( m , 1h ), 1 . 68 - 1 . 89 ( m , 4h ), 1 . 42 ( s , 9h ), 1 . 35 ( s , 3h ), 1 . 23 ( s , 3h ), 1 . 04 ( s , 9h ); 13 c nmr ( cdcl 3 , 75 mhz ) δ 162 . 8 , 156 . 4 , 150 . 8 , 140 . 7 , 135 . 7 , 135 . 6 , 133 . 5 , 133 . 3 , 130 . 1 , 125 . 1 , 128 . 0 , 115 . 5 , 108 . 8 , 86 . 9 , 79 . 8 , 76 . 7 , 74 . 3 , 64 . 7 , 47 . 4 , 28 . 6 , 28 . 4 , 27 . 1 , 25 . 8 , 19 . 5 . a mixture of compound 14 ( 770 mg , 1 . 07 mmol ) and ac 2 o ( 0 . 15 ml , 1 . 59 mmol ) in pyridine ( 10 ml ) was stirred at room temperature overnight and concentrated . the residue was resolved by silica gel column chromatography ( hex / etoac = 1 / 2 ) to give compound 15 ( 720 mg , 0 . 94 mmol , 88 %) as a white foam . 1 h nmr ( cdcl 3 , 300 mhz ) δ 9 . 16 ( brs , 1h ), 7 . 65 - 7 . 68 ( m , 4h ), 7 . 26 - 7 . 47 ( m , 7h ), 5 . 38 ( m , 1h ), 4 . 90 ( dd , 1h , j = 4 . 8 , 10 . 8 ), 4 . 82 ( m , 1h ), 3 . 99 - 4 . 10 ( m , 3h ), 3 . 58 - 3 . 92 ( m , 4h ), 3 . 30 ( m , 1h ), 2 . 86 ( m , 1h ), 2 . 50 ( m , 1h ), 2 . 08 ( s , 3h ), 1 . 69 - 1 . 94 ( m , 3h ), 1 . 43 ( s , 9h ), 1 . 37 ( s , 3h ), 1 . 25 ( s , 3h ), 1 . 04 ( s , 9h ); 13 c nmr ( cdcl 3 , 75 mhz ) δ 170 . 8 , 162 . 8 , 156 . 0 , 150 . 7 , 149 . 9 , 136 . 4 , 135 . 7 , 135 . 6 , 133 . 5 , 133 . 1 , 130 . 2 , 130 . 1 , 128 . 1 , 128 . 0 , 124 . 0 , 114 . 9 , 108 . 9 , 85 . 1 , 79 . 8 , 74 . 4 , 73 . 9 , 64 . 4 , 47 . 5 , 39 . 8 , 28 . 6 , 28 . 4 , 27 . 0 , 25 . 7 , 21 . 4 , 19 . 5 . to a stirred solution of compound 15 ( 720 mg , 0 . 94 mmol ) in thf ( 10 ml ) was added et 3 n . 3hf ( 0 . 31 ml , 1 . 89 mmol ). the reaction mixture was stirred at room temperature overnight and concentrated . the residue was resolved by silica gel column chromatography ( ch 2 cl 2 / meoh = 15 / 1 to 10 / 1 ) to give compound 16 ( 440 mg , 0 . 84 mmol , 89 %) as a white solid . 1 h nmr ( dmso - d 6 , 300 mhz ) δ 11 . 37 ( brs , 1h ), 7 . 64 ( s , 1h ), 6 . 81 ( t , 1h , j = 5 . 7 ), 5 . 10 ( m , 1h ), 4 . 89 ( m , 1h ), 4 . 72 ( dd , 1h , j = 5 . 1 , 10 . 5 ), 4 . 00 - 4 . 10 ( m , 2h ), 4 . 87 ( m , 1h ), 3 . 19 - 3 . 56 ( m , 4h ), 2 . 87 - 3 . 10 ( m , 2h ), 2 . 10 ( dd , 1h , j = 5 . 4 , 13 . 5 ), 2 . 03 ( s , 3h ), 1 . 91 - 1 . 99 ( m , 1h ), 1 . 66 - 1 . 81 ( m , 2h ), 1 . 33 ( s , 9h ), 1 . 32 ( s , 3h ), 1 . 22 ( s , 3h ); 13 c nmr ( dmso - d 6 , 75 mhz ) δ 170 . 9 , 163 . 7 , 156 . 3 , 151 . 2 , 143 . 1 , 112 . 8 , 108 . 1 , 85 . 3 , 78 . 6 , 77 . 0 , 76 . 1 , 74 . 8 , 62 . 5 , 46 . 8 , 38 . 6 , 28 . 8 , 28 . 5 , 26 . 3 , 21 . 6 . to a solution of compound 16 ( 430 mg , 0 . 82 mmol ) in pyridine ( 3 . 3 ml ) and dioxane ( 2 . 8 ml ) was added a solution of 2 - chloro - 4h - 1 , 3 , 2 - benzodioxaphosphorin - 4 - one ( 213 mg , 1 . 15 mmol ) in dioxane ( 2 . 1 ml ) at room temperature . after 20 min a mixture of tributylammonium pyrophosphate in dmf ( 0 . 2 m , 11 . 5 ml , 2 . 3 mmol ) and tributylamine ( 1 . 3 ml , 5 . 2 mmol ) was added . after 20 min a solution of iodine ( 295 mg , 1 . 15 mmol ) and water ( 0 . 46 ml ) in pyridine ( 23 ml ) was added . after 30 min the reaction was quenched by the addition of aqueous na 2 so 3 ( 5 %, 0 . 5 ml ). the solvents were removed in vacuo . the residue was treated with nh 4 oh ( conc ., 40 ml ) for 4 h at room temperature and the mixture was lyophilized . the residue was dissolved in water ( 45 ml ), and the mixture was filtered ( 0 . 2 μm ). purification by ion - exchange hplc ( dionex biolc dnapac pa - 100 , 22 × 250 mm , eluent a = water , eluent b = 1 m aq . nh 4 hco 3 , constant a for 5 min , then gradient from 0 to 50 % b in 25 min , flow rate = 10 ml / min , r t = 12 - 15 min ) gave 3 ′- oh triphosphate ( 180 μmol , 22 %) as a colorless foam after lyophilization . 1 h - nmr ( d 2 o , 300 mhz ): δ 7 . 61 ( s , 1h ), 4 . 88 ( m , 1h ), 4 . 35 ( m , 1h ), 4 . 17 ( m , 1h ), 4 . 09 ( m , 1h ), 3 . 78 - 3 . 95 ( m , 5h ), 3 . 07 ( m , 1h ), 2 . 95 ( m , 1h ), 1 . 98 - 2 . 07 ( m , 2h ), 1 . 78 ( m , 1h ), 1 . 28 ( s , 3h ), 1 . 24 ( s , 9h ), 1 . 18 ( s , 3h ); 31 p - nmr ( d 2 o , 120 mhz ): δ − 6 . 1 ( d , p ); − 10 . 1 ( d , 1p ); − 21 . 6 ( t , 1p ). to a 10 mm solution of 3 ′- oh triphosphate ( 5 ml , 50 mmol ) was added dowex 50wx2 . the reaction mixture was heated at 55 ° c . for 2 h , diluted with water ( 20 ml ) and filtered ( 0 . 2 μm ). purification by reverse phase hplc ( sunfire prep c 18 column , sum , 30 × 250 mm , eluent a = 25 mm teaa ph 7 , eluent b = 50 % ch 3 cn in a , gradient from 0 to 100 % b in 30 min , flow rate = 10 ml / min , r t = 14 min ) gave compound 17 ( 30 μmol ) as a colorless foam after lyophilization . 1 h - nmr ( d 2 o , 300 mhz ): δ 7 . 68 ( s , 1h ), 4 . 95 ( m , 1h ), 4 . 38 ( m , 1h ), 3 . 64 - 3 . 98 ( m , 6h ), 3 . 48 ( m , 1h ), 2 . 81 - 2 . 89 ( m , 2h ), 2 . 13 ( m , 1h ), 1 . 86 - 1 . 96 ( m , 2h ), 1 . 65 ( m , 1h ); 31 p - nmr ( d 2 o , 120 mhz ): δ − 8 . 3 ( d , p ); − 10 . 2 ( d , 1p ); − 21 . 9 ( t , 1p ). a solution of compound 17 ( 8 mmol ) in aqueous k 2 hpo 4 ( 0 . 5 m , 0 . 5 ml ) was mixed with a solution of bodipy 576 / 589 - osu ( 5 mg ) in dmso ( 0 . 6 ml ) and acetone ( 0 . 3 ml ). the mixture was incubated at rt in the dark for 2 h . the mixture was diluted with water ( 15 ml ) and resolved by ion - exchange hplc ( dionex biolc dnapac pa - 100 , 22 × 250 mm , eluent a = water , eluent b = 1 m aq . nh 4 hco 3 , c = ch 3 cn , gradient from 80 % a / 20 % c to 40 % a / 40 % b / 20 % c in 30 min , flow rate = 10 ml / min , r t = 16 mm ), to give compound 18 . the diol linker is cleavable by periodate within seconds at room temperature . the remaining aldehyde can form an imine which is activated towards base catalyzed beta - elimination under sufficiently mild conditions , with the nucleobase acting as the leaving group . to develop the reagents for elimination of scar triphosphate - aldehyde which was obtained from the treatment of compound 17 with 10 mm naio 4 was used as model compound . a variety of conditions were explored that might eliminate the scar and the reaction was analyzed by ion - exchange hplc ( buffer a : water ; buffer b : 1m nh 4 hco 3 ; gradient 0 ˜ 35 % b over 20 min ). % product conditions ph 5 min 10 min 20 min 30 min a 11 . 0 18 52 92 10 . 0 35 70 87 9 . 0 13 20 30 b 11 . 2 40 80 94 10 . 5 50 92 99 10 . 0 70 98 9 . 0 73 98 c 11 . 0 99 99 10 . 0 90 99 9 . 0 32 67 conditions : a : 100 mm glycine , 55 ° c . b : 500 mm morpholine , 55 ° c . c : 100 mm pyrrolidine , 55 ° c . thirteen dna polymerases and reverse transcriptases performing well last time were tested again for their abilities to incorporate the triphosphate carrying a bodipy 576 / 589 fluor ( fig6 ). the incubation time was extended to 30 min to see the boundaries for the enzymes to still consume all the primer s without leading to n + 2 bands . the result ( see next page ) showed that most of the polymerases and reverse transcriptases might achieve this goal even if the incubation time was extended to 30 mm . here only trace amount of n + 2 products were seen with deep vent ( exo -) and bst dna polymerases . gamma - 32 p - labeled primer ( 2 . 5 pmol ), cold primer ( 22 . 5 pmol ) and template ( 30 pmol ) were annealed by incubation at 95 ° c . for 5 min in various reaction buffer and slowly cooled to room temperature , followed by the addition of various dna polymerase and reverse transcriptases . the extension was initiated by adding mjk &# 39 ; s nucleoside triphosphates ( mj - 008 - 065 ) ( final concentration of 100 μm ). after 30 min , the reaction was quenched by 10 mm edta in formamide loading buffer ( 20 μl ); products were resolved by 14 % page . cgc att atg ctg agt gat acc tgc aat gtg ctt ctt ctg - 5 ′ template aa ( dna template ) following a literature procedure [ wellington et al , 2009 ], see also [ bhattacharya et al , 1995 ], a mixture of pd ( oac ) 2 ( 36 mg , 0 . 16 mmol ) and asph 3 ( 98 mg , 0 . 32 mmol ) in anhydrous dmf ( 10 ml ) is stirred at rt for 30 min . it is added to a mixture of glycal 1 ( 280 mg , 0 . 80 mmol ), 5 - iodoheterocycle 2 ( 200 mg , 0 . 80 mmol ), and tributylamine ( 0 . 28 ml , 0 . 12 mmol ) in dmf ( 10 ml ). the resulting mixture is stirred at 60 ° c . overnight . after cooling , the mixture is treated with acetic acid ( 0 . 2 ml ) and tbaf ( 1m in thf , 2 ml ) and stirred at rt for 1 h . volatiles are removed by rotary evaporation under reduced pressure . the product is purified from the residue by flash chromatography ( silica , gradient ch 2 cl 2 : meoh = 15 : 1 to 10 : 1 ). the appropriate fraction ( by tlc ) is collected , evaporated and dissolved in acetic acid / acetonitrile ( 7 ml / 7 ml ). to this solution is added nabh ( oac ) 3 ( 370 mg , 1 . 75 mmol ) at 0 ° c . and stirred for 2 h . volatiles are removed by rotary evaporation under reduced pressure . the product is purified from the residue by flash chromatography ( silica , gradient ch 2 cl 2 : meoh = 7 : 1 to 4 : 1 ) to give a white solid ( 100 mg , 51 %). the 9 - deazapurine skeleton is prepared by the method of liu et al . [ liu et al , 2005 ], which is incorporated in its entirety herein by reference . the heterocycle and various corresponding nucleosides for the 8 - aza - 9 - deaza purine skeleton implementation of the adenine analog for the second architecture is well known from the natural product formycin , as are modified formycins carrying a tag on the n7 position ( purine numbering ). the following references are incorporated in their entirety by citation [ rosemeyer et al 1997 ][ muehlegger et al , 1996 ][ muhlegger et al , 2001 ][ seela et al , 1996 ]. the formycin analogs for guanosine nucleosides are well known , as are modified heterocycles carrying a tag on the n7 position ( purine numbering ). the following reference is incorporated in its entirety by citation [ sanghvi et al , 1991 ]. the aldehyde (˜ 3 mg ) in fig7 , prepared by the periodate cleavage of the corresponding diol , 1 was incubated under the following conditions . the reaction was monitored by reverse hplc ( c 18 column , buffer a : 25 mm teaa , ph 7 ; buffer b : acetonitrile ; gradient : 0 - 40 % b over 30 minutes ). conditions : ( a - 1 ) ph 11 ( 0 . 1 m nahco 3 ), rt ; ( a - 2 ) ph 11 , 37 ° c . ; ( a - 3 ) ph 11 , 55 ° c . ( b - 1 ) ph 11 . 8 ( 0 . 1 m caps ), rt ; ( b - 2 ) ph 11 . 8 , 37 ° c . ; ( b - 3 ) ph 11 . 8 , 55 ° c . the reaction was monitored after 10 mm , 20 mm , 30 min , 1 h , and 2 h , but the reaction was not completed after 2 h incubation . from these results , the cleavage reaction can be done in ph 11 at 55 ° c . in less than 20 min and also in ph 11 . 8 at 37 or 55 ° c . in 1020 min . under this condition , the reaction was faster than condition a - 1 , but it took more than 7 h to complete the reaction . with an amine as a catalyst , the following rates of cleavage were measured . metzker , m . l . 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