to a mixture of compound 7 ( 183 
 g , 279 mmol ) and imidazole ( 34.4 g , 505 mmol ) in dichloromethane 
 ( 2 l ) was added tbdpscl ( 72.2 ml , 278 mmol ) . the mixture was stirred 
 at room temperature for 1 h and washed with h2o , dried 
 over na2so4 , and filtered . the solution was 
 concentrated by rotary evaporation , and the residue was resolved by 
 flash chromatography ( silica , ethyl acetate / hexanes = 1:3 to 1:2 ) . the residue was then redissolved in dichloromethane 
 ( 2l ) , to which were then added dmap ( 1.5 g ) , triethylamine ( 130 ml ) , 
 and di - tert - butyl dicarbonate ( 120 g ) . the mixture 
 was stirred at room temperature overnight and washed with brine , dried 
 over na2so4 , and filtered . the solution was 
 concentrated by rotary evaporation , and the residue was resolved by 
 flash chromatography ( silica , ethyl acetate / hexanes = 1:4 to 1:3 ) 
 to give 8 as a pale yellow solid ( 235 g , 77% ) : mp 5658 
 c ; h nmr ( 300 mhz , cdcl3 )  8.40 
 ( s , 1h ) , 8.01 ( d , 2h , j = 8.7 hz ) , 7.627.76 
 ( m , 8h ) , 7.267.49 ( m , 12h ) , 7.19 ( d , 2h , j = 8.4 hz ) , 5.59 ( d , 1h , j = 3.0 hz ) , 4.83 ( m , 1h ) , 
 4.354.58 ( m , 3h ) , 3.854.10 ( m , 2h ) , 2.853.05 
 ( m , 2h ) , 1.38 ( s , 18h ) , 1.04 ( s , 9h ) , 0.98 ( s , 9h ) ; c 
 nmr ( 75 mhz , cdcl3 )  160.6 , 151.5 , 150.1 , 147.0 , 
 145.7 , 143.2 , 136.5 , 135.9 , 135.8 , 135.6 , 134.4 , 133.4 , 133.3 , 131.5 , 
 131.4 , 130.6 , 129.9 , 128.2 , 128.1 , 127.9 , 127.9 , 126.7 , 123.9 , 101.3 , 
 84.5 , 84.0 , 78.1 , 67.4 , 65.2 , 35.1 , 28.0 , 27.0 , 26.5 , 19.43 , 19.39 ; 
 hrms ( tof- esi ) m / z calcd for c60h70n4o12si2na 
 ( m + na ) 1117.4421 , found 1117.4405 . to a solution of the compound 8 ( 65 g , 59.3 mmol ) in thf ( 700 ml ) was added diborane in thf ( 1 m , 
 200 ml , 3.4 equiv ) at 0 c . it was quenched with 70 
 ml of h2o for 5 min and treated with sodium perborate ( 100 
 g ) in water ( 600 ml ) . the mixture was vigorously stirred at room temperature 
 for 1 h. thf was removed by rotary evaporation , and the residue was 
 extracted with ethyl acetate . the organic layer was then dried over 
 na2so4 and filtered . the solution was concentrated 
 by rotary evaporation , and the residue was resolved by flash chromatography 
 ( silica , ethyl acetate / hexanes = 1:4 to 1:3 ) to give 9 as a pale yellow solid ( 61 g , 92% ) : mp 7173 c ; h nmr ( 300 mhz , cdcl3 )  8.62 ( s , 1h ) , 8.10 
 ( d , 2h , j = 8.7 hz ) , 7.647.74 ( m , 4h ) , 7.147.50 
 ( m , 18h ) , 4.68 ( s , 1h ) , 4.60 ( t , 2h , j = 6.3 hz ) , 
 4.004.28 ( m , 4h ) , 3.64 ( br s , 1h ) , 3.09 ( t , 2h , j = 5.4 hz ) , 1.42 ( s , 18h ) , 1.09 ( s , 9h ) , 0.81 ( s , 9h ) ; c nmr ( 75 mhz , cdcl3 )  160.2 , 150.3 , 147.1 , 145.6 , 
 143.3 , 136.7 , 136.0 , 135.9 , 135.8 , 134.2 , 133.9 , 133.7 , 133.4 , 132.0 , 
 130.2 , 129.95 , 129.86 , 128.1 , 127.9 , 125.0 , 124.0 , 84.2 , 83.7 , 81.9 , 
 79.4 , 67.3 , 62.8 , 35.0 , 28.1 , 27.2 , 26.9 , 19.5 , 19.2 ; hrms ( tof - esi ) m / z calcd for c60h72n4o13si2cl ( m + cl ) 1147.4328 , found 1147.4365 ; c62h75n4o15si2 ( m + ch3coo ) 1171.4773 , found 1171.4825 . a mixture of compound 9 ( 128 
 g , 115 mmol ) , acetic anhydride ( 20 ml ) , pyridine ( 30 ml ) , and dmap 
 ( 0.2 g ) in dichloromethane ( 1200 ml ) was stirred at room temperature 
 for 2 h. the mixture was washed with 0.5 n hcl solution and brine . the residue 
 was purified by flash chromatography ( silica , ethyl acetate / hexanes 
 = 1:4 ) to give a light yellow solid ( 128 g , 96% ) : mp 7476 
 c ; h nmr ( 300 mhz , cdcl3 )  8.70 
 ( s , 1h ) , 8.15 ( d , 2h , j = 8.7 hz ) , 7.557.75 
 ( m , 4h ) , 7.157.45 ( m , 18h ) , 4.99 ( s , 1h ) , 4.86 ( s , 1h ) , 4.454.65 
 ( m , 2h ) , 4.054.2 ( m , 3h ) , 3.81 ( dd , 1h , j = 10.2 , 3.3 hz ) , 3.05 ( t , 2h , j = 6.9 hz ) , 1.90 
 ( s , 3h ) , 1.41 ( s , 18h ) , 1.07 ( s , 9h ) , 0.79 ( s , 9h ) ; c 
 nmr ( 75 mhz , cdcl3 )  168.7 , 160.2 , 150.2 , 147.1 , 
 145.6 , 143.7 , 136.6 , 136.1 , 135.9 , 135.8 , 134.7 , 133.6 , 133.4 , 132.7 , 
 132.0 , 130.1 , 130.0 , 129.9 , 129.8 , 127.9 , 124.2 , 124.0 , 84.4 , 84.1 , 
 81.6 , 79.4 , 77.1 , 67.3 , 63.3 , 34.8 , 28.1 , 27.1 , 27.0 , 21.0 , 19.4 , 
 19.2 ; hrms ( tof - esi ) m / z calcd for 
 c62h74n4o14si2na ( m + na ) 1177.4632 , found 1177.4620 . to a solution of compound 10 ( 128 g , 110.8 mmol ) in dichloromethane ( 1100 ml ) was added 
 trifluoroacetic acid ( 33 ml ) at room temperature . the 
 mixture was stirred for 3 h , neutralized by aqueous nahco3 , and mixed with water and extracted with dichloromethane . the residue was then resolved by flash 
 chromatography ( silica , ethyl acetate / hexanes = 1:2 ) to give the nh2 ( boc removed ) product as a pale yellow solid ; h nmr ( 300 mhz , cdcl3 )  8.62 ( d , 1h , j = 0.9 hz ) , 8.15 ( d , 2h , j = 8.7 hz ) , 7.557.7 
 ( m , 4h ) , 7.17.5 ( m , 18h ) , 5.02 ( s , 1h ) , 4.77 ( s , 1h ) , 4.44.55 
 ( m , 2h ) , 4.054.15 ( m , 3h ) , 3.86 ( dd , 1h , j = 13.8 , 7.2 hz ) , 3.04 ( t , 2h , j = 6.8 hz ) , 1.89 
 ( s , 3h ) , 1.07 ( s , 9h ) , 0.79 ( s , 9h ) ; c nmr ( 75 mhz , cdcl3 )  168.9 , 162.3 , 153.1 , 147.1 , 145.9 , 136.2 , 136.1 , 
 135.9 , 135.8 , 135.4 , 133.7 , 133.5 , 133.1 , 132.1 , 130.0 , 129.9 , 129.8 , 
 128.0 , 127.9 , 127.7 , 124.0 , 122.8 , 114.4 , 84.1 , 81.8 , 79.4 , 77.4 , 
 66.7 , 63.4 , 34.9 , 27.2 , 26.9 , 21.1 , 19.4 , 19.2 . dmap ( 4 g ) and acetic anhydride 
 ( 60 ml ) were added to the solution , and the mixture was stirred for 
 5 h at 60 c . pyridine was then removed by rotary evaporation , 
 and the residue was dissolved in ethyl acetate . the organic phase was separated , 
 dried , and purified by column chromatography ( silica gel , ethyl acetate / hexanes ) 
 to give a pale yellow solid , which was a mixture of the n - acetyl and n , n - diacetyl compounds . spectral data for n - acetyl compound : h nmr ( 300 mhz , cdcl3 )  10.64 ( s , 1h ) , 8.72 ( d , 
 1h , j = 0.9 hz ) , 8.15 ( d , 2h , j = 
 8.7 hz ) , 7.67.7 ( m , 4h ) , 7.17.5 ( m , 18h ) , 5.03 ( s , 
 1h ) , 4.83 ( s , 1h ) , 4.554.65 ( m , 2h ) , 4.054.2 ( m , 3h ) , 
 3.85 ( dd , 1h , j = 10.2 , 3.3 hz ) , 3.12 ( t , 2h , j = 6.9 hz ) , 2.45 ( s , 3h ) , 1.92 ( s , 3h ) , 1.08 ( s , 9h ) , 0.76 
 ( s , 9h ) . the mixture of n - acetyl and n , n - diacetyl compounds was mixed with triethylamine 
 trihydrofluoride ( 160 ml ) , triethylamine ( 140 ml ) , and 
 thf ( 1400 ml ) then stirred at room temperature for 24 h. the mixture 
 was poured into aqueous nahco3 and extracted with ethyl 
 acetate . the residue was purified by 
 flash chromatography ( silica , ethyl acetate / hexanes = 3:1 to ethyl 
 acetate 100% ) to give the product as a pale yellow solid ( 49 g , this 
 is a mixture of mono- and diacetyl compounds and the monoacetyl is 
 major , 84% ) : mp 5254 c ; h nmr ( 300 mhz , 
 cdcl3 )  10.52 ( s , 1h ) , 8.73 ( d , 1h , j = 0.6 hz ) , 8.16 ( d , 2h , j = 8.7 hz ) , 7.43 ( d , 2h , j = 8.7 hz ) , 5.03 ( dd , 1h , j = 3.0 , 1.8 
 hz ) , 4.91 ( dd , 1h , j = 2.7 , 0.9 hz ) , 4.64.8 
 ( m , 2h ) , 4.3 ( m , 1h ) , 4.054.2 ( m , 3h ) , 3.93.95 ( m , 
 1h ) , 3.22 ( t , 2h , j = 6.9 hz ) , 2.953.05 ( m , 
 1h ) , 2.44 ( s , 3h ) , 2.06 ( s , 3h ) ; c nmr ( 75 mhz , cdcl3 )  170.2 , 169.3 , 161.6 , 147.1 , 145.6 , 145.5 , 135.9 , 
 130.0 , 126.1 , 124.0 , 118.2 , 83.5 , 81.0 , 78.5 , 77.4 , 68.1 , 61.5 , 34.9 , 
 26.5 , 21.1 ; hrms ( tof - esi ) m / z calcd 
 for c22h24n4o11na ( m + 
 na ) 543.1334 , found 543.1331 . a mixture of compound 11 ( 23.4 
 g , 45.0 mmol ) , trityl chloride ( 13.8 g , 49.5 mmol ) , dmap ( 0.4 g ) in 
 triethylamine ( 12.5 ml ) , and dichloromethane ( 350 ml ) was stirred 
 at room temperature for 2 h. it was washed with water , and the organic 
 layer was dried over na2so4 , filtered , and concentrated . the residue was purified by flash chromatography ( silica , ethyl acetate / hexanes 
 = 2:1 ) to give a light yellow solid : h nmr ( 300 mhz , cdcl3 )  10.59 ( s , 1h ) , 8.73 ( d , 1h , j = 
 0.6 hz ) , 8.16 ( d , 2h , j = 8.7 hz ) , 7.27.5 
 ( m , 17h ) , 5.08 ( dd , 1h , j = 2.1 , 0.9 hz ) , 4.92 ( dd , 
 1h , j = 2.1 , 0.9 hz ) , 4.64.8 ( m , 2h ) , 4.154.3 
 ( m , 2h ) , 3.65 ( d , 2h , j = 4.5 hz ) , 3.22 ( t , 2h , j = 3.9 hz ) , 3.08 ( d , 1h , j = 3.0 hz ) , 
 2.44 ( s , 3h ) , 2.07 ( s , 3h ) ; c nmr ( 75 mhz , cdcl3 )  169.6 , 168.9 , 161.4 , 147.1 , 145.6 , 145.5 , 143.3 , 135.7 , 
 130.0 , 128.7 , 128.3 , 127.6 , 126.1 , 124.0 , 118.4 , 87.9 , 82.7 , 80.9 , 
 79.2 , 79.9 , 68.0 , 62.2 , 35.0 , 26.5 , 21.1 . the product was then 
 dissolved in dichloromethane ( 350 ml ) and treated with dess - martin 
 periodinane ( 28 g , 66 mmol ) at room temperature and stirred for 4 
 h. it was filtered through celite , and the filtrate was concentrated . the residue was purified by flash chromatography ( silica , ethyl acetate / hexanes 
 = 1:1 ) to give a light yellow solid . it was then dissolved in dichloromethane 
 ( 600 ml ) and treated with trifluoroacetic acid ( 30 ml ) 
 and stirred at room temperature for 3 h. the mixture was neutralized 
 by aqueous sodium bicarbonate and mixed with water and extracted with 
 dichloromethane . the residue 
 was purified by flash chromatography ( silica , ethyl acetate / hexanes 
 = 2:1 ) to give a light yellow solid ( 15.9 g , 68% ) : mp 6668 
 c ; h nmr ( 300 mhz , cdcl3 )  10.64 
 ( s , 1h ) , 8.65 ( s , 1h ) , 8.17 ( d , 2h , j = 9.0 hz ) , 
 7.47 ( d , 2h , j = 8.7 hz ) , 5.22 ( d , 1h , j = 9.6 hz ) , 5.09 ( d , 1h , j = 9.3 hz ) , 4.64.9 
 ( m , 2h ) , 4.25 ( t , 1h , j = 2.7 hz ) , 3.98 ( m , 2h ) , 
 3.26 ( t , 2h , j = 6.9 hz ) , 2.44 ( s , 3h ) , 2.07 ( s , 
 3h ) ; c nmr ( 75 mhz , cdcl3 )  207.5 , 
 169.6 , 168.4 , 162.8 , 147.3 , 146.5 , 145.2 , 137.3 , 130.1 , 125.7 , 124.1 , 
 115.5 , 81.4 , 76.4 , 75.0 , 68.7 , 61.6 , 35.0 , 26.6 , 20.5 ; hrms ( tof - esi ) m / z calcd for c22h22n4o11na ( m + na ) 541.1177 , found 
 541.1181 . to a solution of compound 12 ( 29.0 g , 51.7 mmol ) in acetic acid ( 200 ml ) and acetonitrile ( 200 
 ml ) was added sodium triacetoxyborohydride ( 21.9 
 g , 103 mmol ) at 0 c and stirred for 1 h. it was poured into 
 water and extracted with ethyl acetate . the combined organic layers 
 were dried over na2so4 , filtered , and concentrated . the residue was resolved by flash chromatography ( silica , ethyl acetate / hexanes 
 = 2:1 to 100% ethyl acetate ) to give 13a/13b as a pale 
 yellow solid ( 20.5 g , 70% ) : mp 164165 c ; hrms ( tof - esi ) m / z calcd for c22h24n4o11na ( m + na ) 543.1334 , found 
 543.1333 . a mixture of compound 13 ( 15 
 g , 28.8 mmol ) and ammonium hydroxide ( 100 ml ) in methanol ( 500 ml ) 
 was stirred at room temperature overnight . the solution was then concentrated 
 in vacuo , and the residue was resolved by flash chromatography ( silica , 
 ethyl acetate / methanol = 15:1 ) to give a light yellow solid ( 11.3 
 g , 90% for two steps ) : mp 155156 c ; h nmr 
 ( 300 mhz , cd3od )  8.52 ( d , 1h , j = 0.6 hz ) , 8.18 ( d , 2h , j = 9.0 hz ) , 7.60 ( d , 2h , j = 8.7 hz ) , 4.82 ( d , 2h , j = 3.6 hz ) , 
 4.64.7 ( m , 2h ) , 3.653.95 ( m , 5h ) , 3.24 ( t , 2h , j = 6.3 hz ) ; c nmr ( 75 mhz , cd3od ) 
  163.6 , 154.0 , 147.0 , 146.8 , 135.2 , 130.3 , 123.4 , 121.7 , 113.5 , 
 83.4 , 79.5 , 75.9 , 70.9 , 67.0 , 61.9 , 34.7 ; hrms ( tof - esi ) m / z calcd for c18h20n4o9na ( m + na ) 459.1122 , found 459.1118 . to a solution of 
 compound 14 ( 30 mg , 0.069 mmol ) in acetonitrile ( 5 ml ) 
 was added dbu ( 0.1 ml ) ; the mixture was then stirred overnight at 
 room temperature . the mixture was then concentrated in vacuo , and 
 the residue was resolved by flash chromatography ( silica , ch2cl2/meoh = 5:1 to 5:2 ) to give a yellow solid ( 15 mg , 
 75% ) : mp 200202 c;h nmr ( 300 mhz , d2o )  8.14 ( s , 1h ) , 4.59 ( d , 1h , j = 
 4.8 hz ) , 4.03 ( t , 1h , j = 4.8 hz ) , 3.96 ( t , 1h , j = 6.0 hz ) , 3.83.9 ( m , 1h ) , 3.72 ( dd , 1h , j = 12.6 , 3.0 hz ) , 3.59 ( dd , 1h , j = 12.6 , 
 4.5 hz ) ; c nmr ( 75 mhz , d2o )  163.3 , 
 150.9 , 135.4 , 116.7 , 115.0 , 83.1 , 80.3 , 74.3 , 70.7 , 61.5 ; hrms ( tof - esi ) m / z calcd for c10h12n3o7 ( m  h ) 286.0681 , 
 found 286.0681 . to a solution of compound 13 ( 500 mg , 0.961 mmol ) , triethylamine ( 0.27 ml ) , and dmap ( 10 mg ) 
 in dichloromethane ( 10 ml ) was added dmtr chloride ( 358 mg , 
 1.057 mmol ) . the mixture was stirred at room temperature for 1 h then 
 washed with water . the residue was purified 
 by flash chromatography ( silica , ethyl acetate ) to give a light yellow 
 solid . this was dissolved in dichloromethane ( 20 ml ) and pyridine 
 ( 0.5 ml ) and treated with acetic anhydride ( 0.18 ml ) and dmap ( 10 
 mg ) . the mixture was stirred at room temperature for 3 h then washed 
 with water . the residue was then resolved 
 by flash chromatography ( silica , ethyl acetate / hexanes = 2:1 ) to give 
 product as a light yellow solid : h nmr ( 300 mhz , cdcl3 )  10.61 ( s , 1h ) , 8.66 ( s , 1h ) , 8.17 ( d , 2h , j = 8.7 hz ) , 7.157.5 ( m , 11h ) , 6.81 ( d , 4h , j = 8.1 hz ) , 5.49 ( t , 1h , j = 4.8 hz ) , 
 5.32 ( dd , 1h , j = 12.3 , 3.6 hz ) , 5.01 ( d , 1h , j = 4.2 hz ) , 4.69 ( t , 2h , j = 6.6 hz ) , 
 4.24.3 ( m , 1h ) , 3.77 ( s , 6h ) , 3.353.45 ( m , 2h ) , 3.21 
 ( t , 2h , j = 6.6 hz ) , 2.44 ( s , 3h ) , 2.06 ( s , 3h ) , 
 2.04 ( s , 3h ) ; c nmr ( 75 mhz , cdcl3 )  
 169.8 , 169.5 , 168.7 , 161.9 , 158.7 , 147.1 , 145.9 , 145.5 , 144.7 , 135.9 , 
 135.8 , 135.7 , 130.3 , 130.1 , 128.3 , 128.1 , 127.1 , 125.9 , 124.0 , 117.0 , 
 113.4 , 86.7 , 80.2 , 77.6 , 74.8 , 71.2 , 68.1 , 62.5 , 60.6 , 55.4 , 35.0 , 
 26.6 , 20.8 , 20.7 . the product was dissolved in dichloromethane 
 ( 30 ml ) and treated with dichloroacetic acid ( 0.6 ml ) . the mixture 
 was stirred at room temperature for 1 h then washed with water . the residue was resolved by flash chromatography 
 ( silica , ethyl acetate / hexanes = 2:1 to ethyl acetate 100% ) to give 16 as a light yellow solid ( 400 mg , 74% ) : mp 160161 
 c ; h nmr ( 300 mhz , cdcl3 )  10.60 
 ( s , 1h ) , 8.73 ( s , 1h ) , 8.17 ( d , 2h , j = 8.7 hz ) , 
 7.47 ( d , 2h , j = 8.7 hz ) , 5.25.4 ( m , 2h ) , 
 4.96 ( d , 1h , j = 4.5 hz ) , 4.74 ( t , 2h , j = 6.3 hz ) , 4.14.2 ( m , 1h ) , 3.954.05 ( m , 1h ) , 3.743.84 
 ( m , 1h ) , 3.26 ( t , 2h , j = 7.2 hz ) , 2.44 ( s , 3h ) , 
 2.23 ( t , 1h , j = 5.7 hz ) , 2.10 ( s , 3h ) , 2.06 ( s , 
 3h ) ; c nmr ( 75 mhz , cdcl3 )  170.3 , 
 169.6 , 168.7 , 162.1 , 147.2 , 146.1 , 145.4 , 136.7 , 130.1 , 125.8 , 124.1 , 
 116.6 , 81.6 , 78.4 , 74.7 , 70.5 , 68.3 , 61.5 , 35.0 , 26.6 , 20.8 ; hrms 
 ( tof - esi ) m / z calcd for c24h26n4o12na ( m + na ) 585.1439 , 
 found 585.1436 . to a solution of compound 16 ( 60 mg , 0.107 mmol ) in pyridine ( 1 ml ) and dioxane ( 2 ml ) was added 
 a solution of 2-chloro-4h-1,3,2-benzodioxaphosphorin-4-one 
 ( 32.4 mg , 0.16 mmol ) in dioxane ( 1.0 ml ) at room temperature . after 
 15 min , a mixture of tributylammonium pyrophosphate 
 in dmf ( 0.2 m , 1.6 ml , 0.32 mmol ) and tributylamine ( 0.17 ml ) 
 was added . after 20 min , a solution of iodine ( 40.6 mg , 0.16 mmol ) 
 and water ( 0.063 ml ) in pyridine ( 3.1 ml ) was added . after 30 min , 
 the reaction was quenched by the addition of aqueous na2so3 ( 5% , until color disappears ) . the residue was dissolved in acetonitrile ( 10 
 ml ) and water ( 10 ml ) and kept at room temperature overnight . it was 
 purified by reverse - phase prep hplc ( 25 mm teaa to 25 mm teaa / ch3cn = 30:70 in 20 min , running time 30 min ) , then the collected 
 fraction was lyophilized ( 12.9 min in the analytical hplc , 50 mm teab 
 to 60% ch3cn + 40% 50 mm teab in 20 min ) . the lyophilized 
 residue was dissolved in ch3cn ( 5 ml ) and treated with 
 dbu ( 0.1 ml ) . the mixture was stirred at room temperature for 24 h. 
 volatiles were removed by rotary evaporation . to the residue was added 
 ammonium hydroxide ( 5 ml ) , and the mixture was stirred at room temperature 
 for 1 h. ammonia was removed by rotary evaporation , and the residue 
 was diluted with water and lyophilized . the residue was dissolved 
 in water , filtered , and resolved by ion exchange hplc ( water to 0.5 
 m ammonium bicarbonate in 25 min ) . the collected fraction was lyophilized 
 to give a yellow solid ( 0.042 mmol , 39% ) : h nmr ( 300 mhz , 
 d2o )  8.17 ( s , 1h ) , 4.04.2 ( m , 6h ) ; p nmr ( 121 mhz , d2o )  7.9 ( d , 1p , j = 20 hz ) , 10.0 ( d , 1p , j = 19 
 hz ) , 21.6 ( t , 1p , j = 19 hz ) ; hrms ( tof - esi ) m / z calcd for c10h15n3o16p3 ( m  h ) 525.9671 , found 525.9666 . an aqueous solution ( 30 ml ) of 15 ( 0.5 
 mg ) was acidified to ph 4 by the addition of dilute aqueous hcl ( 10 
 mm ) . the ph of the solution was then varied by addition of various 
 amounts of dilute aqueous naoh ( 10 or 100 mm ) . the pka was determined by plotting the ph versus the quotient of 
 absorption at two different wavelengths ( 400 nm/380 nm ) . compound 15 was dissolved in a dilute hcl aqueous solution 
 ( ph 2.0 ) and solution of 25 mm et3n  hoac buffer 
 ( ph 7.0 ) . the solutions 
 were incubated at room temperature ( ph 2 ) or 90 c ( ph 7 ) . at 
 time intervals , aliquots ( 10 l ) were removed , neutralized with 
 aqueous triethylammonium bicarbonate buffer ( 50 mm ; ph 8 ; 0.1 
 ml ) , and analyzed by analytical rp - hplc ( sunfire c18 5 m , 3.0 
  150 mm , eluent a = 50 mm teab , eluent b = 35% ch3cn and 65% 50 mm teab , gradient from 100% a to 70% a , 30% b in 30 
 min , flow rate 0.5 ml / min ) . transcription 
 templates were 
 prepared by independently combining equimolar ratios of top strand 
 ( nlt1 ) and bottom strand ( nl standard , nlp1 , nlp2 , nlp3 , or nlp4 ) 
 in 1 transcription buffer ( 20 mm nacl , 40 mm tris ph 7.8 , 6 
 mm mgcl2 , 2 mm spermidine , and 10 mm dtt ) , heating to 95 
 c , and then cooling to room temperature . transcription 
 reactions contained a final concentration of 0.2 m template 
 dna ( nlt1 and various bottom strands as indicated in figure 2 ) , 1 transcription buffer ( 20 mm nacl , 40 
 mm tris ph 7.8 , 6 mm mgcl2 , 2 mm spermidine , and 10 mm 
 dtt ) , 1 ci/l p - gtp , mc t7 rna pol 
 rna polymerase ( 0.05 units/l final ) , and 0.5 mm each rntp in 
 the minus experiment or 0.5 mm each rntp and rztp in the plus experiment . reactions were incubated at 37 c for 40 min and 2 h. reactions 
 were quenched with three - fold formamide quench buffer , and samples 
 were resolved on a 20% page .
rearranging hydrogen bonding groups 
 adds nucleobases to an artificially 
 expanded genetic information system ( aegis ) , pairing orthogonally 
 to standard nucleotides . 
 we report here a large - scale synthesis of 
 the aegis nucleotide carrying 2-amino-3-nitropyridin-6-one ( trivially 
 z ) via heck coupling and a hydroboration / oxidation sequence . 
 riboz is more stable against epimerization than its 2-deoxyribo 
 analogue . 
 further , t7 rna polymerase incorporates ztp opposite its 
 watson  crick complement , imidazo[1,2-a]-1,3,5-triazin-4(8h)one ( trivially p ) , laying grounds for using this  second - generation 
 aegis z : p pair to add amino acids encoded by mrna .