Improved synthesis and polymerase recognition of 7-deaza-7-modified α-l-threofuranosyl guanosine analogs

Threofuranosyl nucleic acid (TNA), an artificial genetic polymer known for its nuclease resistance and acid stability, has grown in popularity as a genetically-encoded material for applications in synthetic biology and biomedicine. TNA oligonucleotide synthesis requires enzymatic or solid phase synthesis pathways that rely on monomer building blocks that are not commercially available and can only be obtained by chemical synthesis. Here we present a synthetic route to 7-deaza-7-modified tGTP and phosphoramidite analogs that is operationally simpler than our previously described strategy. The new methodology offers an HPLC-free route to tGTP analogs that are recognized by engineered TNA polymerases and can be incorporated with continued TNA synthesis.


Advantages Drawbacks
Extensions of classic Ludwig-Eckstein Method

General Information
All moisture sensitive reactions were performed in anhydrous solvents under an argon or nitrogen atmosphere.All commercial reagents, solvents and anhydrous solvents were used without further purification.Reaction progresses were monitored by thin layer chromatography using glass-backed analytical Silica Plate with UV active F254 indicator.Flash column chromatography was performed with Silica Flash® P60 silica gel (40-63 µm particle size) for most of the crude reaction mixture.Yields are reported as isolated yields of pure compounds. 1H, 13 C, and 31 P NMR spectra were analysed on 400 MHz NMR spectrometer (Bruker DRX) at the University of California, Irvine NMR Facility. 1 H values are reported in parts per million relative to Me4Si or corresponding deuterium solvents used as an internal standard. 13C values are reported in parts per million relative to corresponding deuterium solvents used as an internal standards.(245 mg, 3.4956 mmol, 1.8 equiv) in an anhydrous solvent 1:1 MeCN/DCM (13 mL) was added 1.3 equiv of (BnO)2PN( i Pr)2 (850 μL, 2.5246 mmol) under a nitrogen atmosphere.The mixture was stirred at room temperature for 3 h (monitored by TLC).To the reaction mixture was added 30% aqueous H2O2 (0.45 mL, 3.953 mmol, 2.5 equiv) at 0°C, and the resulting mixture was stirred at room temperature for 20 min.
At which the reaction was concentrated under reduced pressure, the crude was diluted with 15-20 times volume of DCM and the organic layer was sequentially washed with saturated NaHCO3 (aq), brine, and water.The combined organic layer was dried with Na2SO4, and evaporated under diminished pressure to obtain crude.The crude residue was purified by liquid loading onto silica with eluents (0%-5% MeOH/DCM) to give pure product (1.45 g, 1.7542 mmol, 90%) as white solid.

7-Deaza-N 2 -pivaloyl-9-(2′-O-benzoyl-α-L-threofuranosyl) guanine 3′-O-monophosphor-2methylimidazolide (9a):
To a solution containing the 8a (600 mg, 0.8301 mmol, 1 equiv) in anhydrous DMF (10 mL) under a nitrogen atmosphere was slowly added anhydrous TEA (0.99 mL, 7.102 mmol, 8.5 equiv) at 0 °C.After 5 min of stirring, 2-methylimidazole (473 mg, 5.761 mmol, 6.9 equiv) was added followed by triphenylphosphine (756 mg, 2.882 mmol, 3.5 equiv).After 10 min of stirring at room temperature, 2,2′-dipyridyl disulfide (635 mg, 2.882 mmol, 3.5 equiv) was added and stirring was continued for an additional 3 h at room temperature with monitoring by analytical HPLC (mobile phase: MeCN/0.05 M TEAA buffer).After consumption of the starting material, the product was precipitated by adding the reaction mixture dropwise with stirring to 300 mL of diethyl ether.The precipitate was collected by centrifuging at 4400 rpm for 5 min at room temperature.The supernatant was discarded, and the pellet was resuspended with minimal amount of DCM.The solution was added dropwise to a premade solution of diethyl ether (300 mL)/TEA (15 mL) containing sodium perchlorate (1.17 g, 9.555 mmol, 11.5 equiv) for a second precipitation.The suspended solid was centrifuged at 4400 rpm for 5 min at room temperature, the supernatant was discarded, and the pellet was washed twice with mixed solvent (20 mL ether) and dried under high vacuum to afford the product 9a (280 mg, 0.4616 mmol, 55%).
The above process was repeated two times and the combined supernatants were evaporated under diminished pressure.The crude was loaded on a silica gel column (packed with 2% H2O/isopropanol containing 1% DIPEA) by liquid loading (2 mL of 2% H2O/isopropanol containing 1% DIPEA) with eluents 5% H2O/isopropanol containing 1% DIPEA and then 2% to 7% H2O in (isopropanol/MeCN 1:1) containing 1% DIPEA.The fractions containing the product were collected and evaporated under diminished pressure at 30-40 °C to afford pure 450 mg fully protected nucleoside triphosphate.The purity was confirmed by its 31 P NMR (see the attached NMR below).
Step 2: A solution of fully protected nucleoside triphosphate 450 mg in 30-33% aqueous NH4OH was stirred for 18 h at 37°C in a sealed tube.After the reaction, the solvent was evaporated under diminished pressure.The solid was resuspended with MilliQ water (5 mL) and the aqueous solution was washed with DCM (10 mL) and EtOAc (10 mL).The organic portion was discarded and the aqueous extract was collected, and evaporated under diminished pressure.The crude solid was resuspended with 2 mL of RNAse free water, filtrated by 0.22 μm syringe filter and dropwise added to the forty times volume of acetone (80 mL) at room temperature containing NaClO4 (850 mg, 6.924 mmol, 15 equiv).The resulting suspension was centrifuged at 4400 rpm for 5 min at room temperature.The supernatant was discarded and the pellet was washed with organic solution (acetone/DCM, 10:1, 2 x 30 mL) to afford the 1a (150 mg, 0.2585 mmol, 56% two-step yield).

7-Deaza-(α-L-threofuranosyl) guanine 3′-diphosphate (11a):
Step 1: To a solution of 8a (200 mg, 0.2767 mmol, 1 equiv) in 0.8 mL of anhydrous DCM was added (BnO)2PN( i Pr)2 (106 μL, 0.3320 mmol, 1.2 equiv) and stirred the reaction for 1-3 h at room temperature under nitrogen atmosphere.After the starting material was consumed, t-BuO2H (5.5 M in C10H22) (125 μL, 0.6917 mmol, 2.5 equiv) was added at 0°C and continued the reaction for another 30 min.The solution was then diluted with 10-20 times volume of DCM and the organic layer was sequentially washed with brine, and water (2x50 mL).The combined organic extracts were dried with Na2SO4, and evaporated under diminished pressure.The crude residue was purified by wet loading over silica gel with eluents MeOH/DCM (1% to 5% containing 1% DIPEA) and the fractions containing the product were collected and evaporated to afford as white solid as DIPEA salt of product (137 mg, 0.1505 mmol, 54%).
Step 2: To the above obtained solid in a round bottom flask was added 50 mg of 10% Pd/C which was evacuated for 20 min and then added 2 mL of anhydrous MeOH.The reaction mixture was stirred in presence of H2 balloon for 12 h at room temperature.The suspension was filtered through celite, washed with MeOH (100 mL) and concentrated to afford the product 11a as white DIPEA salt of diphosphate (110 mg, 0.1505 mmol, 53% two-step yield).

7-Deaza-(α-L-threofuranosyl) guanine 3′-triphosphate (1a):
To a solution of 11a (92 mg, 0.1261 mmol, 1 equiv) in 0.4 mL of anhydrous DCM was added (BnO)2PN( i Pr)2 (48 μL, 0.1505 mmol, 1.2 equiv) and stirred the reaction for 3 h at room temperature under nitrogen atmosphere.After the starting material was consumed, t-BuO2H (5.5 M in C10H22) (57 μL, 0.3152 mmol, 2.5 equiv) was added at 0°C and continued the reaction for another 30 min.The solution was then diluted with 10-20 times volume of DCM and the organic layer was sequentially washed with brine, and water (2 x 50 mL).The combined organic extracts were dried with Na2SO4 and evaporated under diminished pressure.The crude residue was purified by wet loading over silica gel with eluents MeOH/DCM (0%-5%-10%, containing 1% DIPEA).The fractions containing the product were collected and evaporated to afford as white solid (68 mg, 0.0687 mmol, 54%).To the above obtained solid (50 mg, 0.0508 mmol) in a round bottom flask was added of 10% Pd/C (20 mg, 0.4 mass equiv) and evacuated for 15-20 minutes and then added 2 mL of anhydrous MeOH.The reaction mixture was stirred in the presence of H2 balloon for 12 h at room temperature.The resulting suspension was filtered through celite, washed with MeOH (100 mL) and concentrated under reduce pressure, dried under high vacuum to afford the product as white solid.The resulting solids were subsequently dissolved in 30-33% aqueous NH4OH (5.0 mL) and stirred for 18 h at 37°C in a sealed tube.After concentrating the solution under reduced pressure in rotavapor, the resulting solid was resuspended in minimum volume of MilliQ water (5 mL) and the solution extracted with DCM (20 mL).
The aqueous layer was concentrated under diminished pressure.To the concentrated aqueous extract was added NaClO4 (92 mg, 0.762 mmol, 15 equiv) in acetone (100 mL) drop wise at room temperature.
The resulting suspension was centrifuged at 4400 rpm for 5 min at room temperature, the supernatant discarded, and the pellet washed with organic solution (10:1 acetone/DCM, 20 mL), and then dried under vacuum to afford the desired product 1a as white solid (29 mg, 0.0503 mmol, 53% yield over three steps, Na salt).