Supporting Information. Solid-Phase Synthesis of Symmetrical 5,5 -Dinucleoside Mono-, Di-, Tri-, and Tetraphosphodiesters

Transcription

1 Supporting Information S1 Solid-Phase Synthesis of Symmetrical 5,5 -Dinucleoside Mono-, Di-, Tri-, and Tetraphosphodiesters Yousef Ahmadibeni and Keykavous Parang* Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, The University of Rhode Island, Kingston, Rhode Island, *Corresponding author. 41 Lower College Road, Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of Rhode Island, Kingston, Rhode Island, 02881, USA; Tel.: ; Fax: ; address:

2 S2 Table of Contents: Page 1. General information. S3 2. Preparation of bis(diisopropylamino)chlorophosphine (1). S3 3. Preparation of [N,N-diisopropylchlorophosphoramidite]-O-[(2-cyanoethyl)-N,Ndiisopropylphosphoramidite] (5). S4 4. Preparation of O-[N,N-diisopropylchlorophosphoramidite]-O-[(2-cyanoethyl)-N,Ndiisopropylphosphoramidite]-2-cyanoethylphosphite (9). S5 5. Preparation of O-[O-[N,N-diisopropylchlorophosphoramidite]-2-cyanoethylphosphite]-O-[(2- cyanoethyl)-n,n-diisopropylphosphoramidite]-2-cyanoethylphosphite (12). S6 6. Preparation of polymer-bound mono-, di-, tri-, and tetraphosphitylating reagents (14 17). S7 7. Solid-phase synthesis of symmetrical 5,5 -dinucleoside mono-, di-, tri-, and tetraphosphodiesters (31 34a e) using polymer-bound mono-, di-, tri-, and tetraphosphitylating reagents S H NMR, 13 C NMR, and 31 P NMR spectra of hydroxyphosphoramidite form of mono-, di-, tri-, and tetraphosphitylating reagents (1, 5, 9, 12 ) and symmetrical 5,5 -dinucleoside mono-, di, tri-, and tetraphosphodiesters (31 34a e). S19 9. Analytical HPLC Profiles of Final Compounds S55

3 S3 1. General Information. All solid-phase reactions were carried out in Bio-Rad polypropylene columns by shaking and mixing using Glass-Col small tube rotator in dry conditions at room temperature unless otherwise stated. Real-time monitoring of loading of compounds on resin beads was carried out with a Thermo-Nicolet 380 FT-IR spectrophotometer using OMNIC software. The chemical structures of final desalted products were characterized by nuclear magnetic resonance spectrometry ( 1 H NMR, 13 C NMR, 31 P NMR) determined on a Bruker NMR spectrometer (400 MHz). 13 C NMR spectra are fully decoupled. Chemical shifts are reported in parts per millions (ppm). The chemical structures of final products were confirmed by a high-resolution PE Biosystems Mariner API time-of-flight electrospray mass spectrometer and quantitative phosphorus analysis. The substitution of the resins for each step was estimated from the weight gain of the resin. Total isolated yields for final products were calculated based on the loading of aminomethyl polystyrene resin-bound mono-, di-, tri-, and tetraphosphitylating reagents and the amount of symmetrical 5-5 -dinucleoside mono-, di-, tri-, and tetraphosphodiesters products. The synthesis of phosphitylating reagents 1, 5, 9, and 12 was carried out under extremely dry conditions and nitrogen. The polymer-bound p-acetoxybenzyl alcohol (13) was synthesized according to the previously reported procedure Preparation of bis(diisopropylamino)chlorophosphine (1). Phosphorus trichloride (875 µl, 10 mmol) and N,N-diisopropylethylamine (DIEA, 3.5 ml, 20 mmol) were added to anhydrous THF (35 ml). Diisopropylamine (2.8 ml, 20 mmol) was added dropwise in 10 min to the solution and the mixture was stirred for 65 min at 0 C to yield 1. The reaction mixture containing 1 was immediately used in the coupling reaction with swelled solution of polymer-bound p-acetoxybenzyl alcohol 13 (2.50 mmol) in THF in the presence of DIEA (10 mmol) as described later. A small amount of 1 was converted to the hydroxyphosphoramidite form (1 ) using water (1 equiv) in the presence of DIEA (1 equiv). The precipitate was filtered off under nitrogen and the solvent was evaporated in vacuum to afford 1 (76%). The chemical structure of the hydroxyphosphoramidite form (1 ) was confirmed by 1 H NMR, 13 C NMR, 31 P NMR, and high-resolution ESI mass spectrometry. Further stability studies on 1 using high-resolution

4 time-of-flight electrospray mass spectrometry showed that the compound remained stable after 2 S4 months storage at 20 C in THF. 1 H NMR (DMSO-d 6, 400 MHz, δ ppm): 0.92 (d, J = 8.0 Hz, CH 3, 24H), (Heptet, J = 8.0 Hz, CH, 4H), (br s, POH, 1H); 13 C NMR (DMSO-d 6, 100 MHz, δ ppm): δ 22.1 (CH 3 ), 43.4 (CHN); 31 P NMR (in DMSO-d 6 and H 3 PO 4 85% in water as external standard, 162 MHz, δ ppm): (s); HR- MS (ESI-TOF) (m/z): calcd, ; found, [M + 1] Preparation of [N,N-diisopropylchlorophosphoramidite]-O-[(2-cyanoethyl)-N,Ndiisopropylphosphoramidite] (5). Phosphorus trichloride (875 µl, 10 mmol) and DIEA (1.75 ml, 10 mmol) were added anhydrous THF (25 ml). Diisopropylamine (1.4 ml, 10 mmol) was added dropwise in 10 min to the solution and the mixture was stirred for 45 min at 0 C to yield diisopropylphosphoramidous dichloride (2). Then 3-hydroxypropionitrile (683 µl, 10 mmol) and DIEA (1.75 ml, 10 mmol) were added dropwise during 10 min period. The stirring was continued for 25 min at 0 C to afford 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (3). Water (180 µl, 10 mmol) and DIEA (1.75 ml, 10 mmol) were added dropwise in 10 min period to the solution mixture. The mixture was stirred for 10 min at 0 C to yield 2-cyanoethyl N,N-diisopropylhydroxyphosphoramidite (4) that was used immediately in the next reaction. Another batch of 2 (1 equiv, 10 mmol) was prepared at the same time in a separate reaction vessel. A mixture of 2 and DIEA (1.75 ml, 10 mmol) was added dropwise in 10 min period to the prepared solution containing 4. The mixture was stirred for 25 min at 0 C to yield 5. The reaction mixture containing diphosphitylating reagent 5 was immediately used in the coupling reaction with the swelled solution of polymer-bound p-acetoxybenzyl alcohol 13 (2.50 mmol) in THF in the presence of DIEA (10 mmol) as described later. A small amount of 5 was converted to the hydroxyphosphoramidite form (5 ) using water (1 equiv) in the presence of DIEA (1 equiv). The precipitate was filtered off under nitrogen and the solvent was evaporated in vacuum to afford 5 (91%). The chemical structure of the hydroxyphosphoramidite form (5 ) was confirmed by 1 H NMR, 13 C NMR,

5 S5 31 P NMR, and high-resolution ESI mass spectrometry. Further stability studies on 5 using highresolution time-of-flight electrospray mass spectrometry showed that the compound remained stable after 2 months storage at 20 C in THF. 1 H NMR (DMSO-d 6, 400 MHz, δ ppm): (m, CH 3, 24H), 2.59 (t, J = 6.0 Hz, CH 2 CN, 2H), (m, CH, 4H), 3.59 (t, J = 6.0 Hz, CH 2 O, 2H), (br s, POH, 1H); 13 C NMR (DMSO-d 6, 100 MHz, δ ppm): δ 21.9 (CH 2 CN), 24.0 (CH 3 ), 24.3 (CH 3 ), 45.3 (CHN), 45.5 (CHN), 57.4 (CH 2 O), (CN); 31 P NMR (in DMSO-d 6 and H 3 PO 4 85% in water as external standard, 162 MHz, δ ppm): (d, J = 18.6 Hz, 1P, CH 2 OPN), (d, J = 18.6 Hz, 1P, HOPN); HR-MS (ESI-TOF) (m/z): calcd, ; found, [M - 1] Preparation of O-[N,N-diisopropylchlorophosphoramidite]-O-[(2-cyanoethyl)-N,Ndiisopropylphosphoramidite]-2-cyanoethylphosphite (9). Phosphorus trichloride (875 µl, 10 mmol) and DIEA (1.75 ml, 10 mmol) were added to anhydrous THF (25 ml). 3-Hydroxypropionitrile (683 µl, 10 mmol) was added dropwise in 10 min to the solution and the mixture was stirred for 25 min at 0 C to yield 2-cyanoethyl phosphorodichloridate (6). Then a mixture of 4 (1 equiv, 10 mmol) and DIEA (1.75 ml, 10 mmol), which was prepared at the same time in a separate reaction vessel, was added dropwise in 10 min period to the solution containing 6. The mixture was stirred for 15 min at 0 C to yield 7. Water (180 µl, 10 mmol) and DIEA (1.75 ml, 10 mmol) were added dropwise in 10 min period to the solution containing 7. The mixture was stirred for 10 min at 0 C to yield 8. Then a mixture of 2 (1 equiv, 10 mmol) and DIEA (1.75 ml, 10 mmol), which was prepared at the same time in a separate reaction vessel, was added dropwise in 10 min period to the solution containing 8. The mixture was stirred for 35 min at 0 C to yield 9. The reaction mixture containing reagent 9 was immediately used in the coupling reaction with swelled solution of polymer-bound p-acetoxybenzyl alcohol 13 (2.50 mmol) in THF in the presence of DIEA (10 mmol) as described later. A small amount of 9 was converted to the hydroxyphosphoramidite form (9 ) using water (1 equiv) in the presence of DIEA (1 equiv). The

6 precipitate was filtered off under nitrogen and the solvent was evaporated in vacuum to afford 9 S6 (84%). The chemical structure of the hydroxyphosphoramidite form (9 ) was confirmed by 1 H NMR, 13 C NMR, 31 P NMR, and high-resolution ESI mass spectrometry. Further stability studies on 9 using highresolution time-of-flight electrospray mass spectrometry showed that the compound remained stable after 2 months storage at 20 C in THF. 1 H NMR (DMSO-d 6, 400 MHz, δ ppm): (m, CH 3, 24H), (dt, J = 6.0 Hz, CH 2 CN, 4H), (m, CH, 4H), (dt, J = 6.0 Hz, CH 2 O, 4H), (br s, POH, 1H); 13 C NMR (DMSO-d 6, 100 MHz, δ ppm): δ 21.8 (CH 2 CN), 22.0 (CH 2 CN), 23.9 (CH 3 ), 24.1 (CH 3 ), 45.1 (CHN), 45.3 (CHN), 57.2 (CH 2 O), 57.4 (CH 2 O), (CN); 31 P NMR (in DMSO-d 6 and H 3 PO 4 85% in water as external standard, 162 MHz, δ ppm): (m, HOPN and CH 2 OPN, 2P), (m, OPOO, 1P); HR-MS (ESI-TOF) (m/z): calcd, ; found, [M - 1] Preparation of O-[O-[N,N-diisopropylchlorophosphoramidite]-2-cyanoethylphosphite]-O-[(2- cyanoethyl)-n,n-diisopropylphosphoramidite]-2-cyanoethylphosphite (12). 2-Cyanoethyl phosphorodichloridate (6, 10 mmol) was prepared as described above. Water (360 µl, 20 mmol) and DIEA (3.5 ml, 20 mmol) were added dropwise in 10 min period to the solution of 6 in anhydrous THF (25 ml) and the mixture was stirred for 10 min at 0 C to yield the intermediate 2-cyanoethyl dihydroxyphosphite (10). Then a mixture of 7 (10 mmol) and DIEA (1.75 ml, 10 mmol), which was prepared at the same time in a separate reaction vessel, was added dropwise in 10 min period to the solution containing 10. The mixture was stirred for 35 min at 0 C to yield 11. Then a mixture of diisopropylphosphoramidous dichloride (2, 1 equiv, 10 mmol) and DIEA (1.75 ml, 10 mmol), which was prepared at the same time in a separate reaction vessel, was added dropwise in 10 min period to the solution containing 11. The mixture was stirred for 35 min at 0 C to yield 12. The reaction mixture containing 12 was immediately used in the coupling reaction with swelled solution of polymer-bound p- acetoxybenzyl alcohol 13 (2.50 mmol) in THF in the presence of DIEA (10 mmol) as described later. A

7 small amount of 12 was converted to the hydroxyphosphoramidite form (12 ) using water (1 equiv) in S7 the presence of DIEA (1 equiv). The precipitate was filtered off under nitrogen and the solvent was evaporated in vacuum to afford 12 (77%). The chemical structure of the hydroxyphosphoramidite form (12 ) was confirmed by 1 H NMR, 13 C NMR, 31 P NMR, and high-resolution ESI mass spectrometry. Further stability studies on 12 using high-resolution time-of-flight electrospray mass spectrometry showed that the compound remained stable after 2 months storage at 20 C in THF. 1 H NMR (DMSO-d 6, 400 MHz, δ ppm): (m, CH 3, 24H), (m, CH 2 CN, 6H), (m, CH, 4H), (m, CH 2 O, 6H); 13 C NMR (DMSO-d 6, 100 MHz, δ ppm): δ 21.9 (CH 2 CN), 22.0 (CH 2 CN), 24.0 (CH 3 ), 24.2 (CH 3 ), 45.2 (CHN), 45.3 (CHN), 57.4 (CH 2 O), 57.5 (CH 2 O), (CN); 31 P NMR (in DMSO-d 6 and H 3 PO 4 85% in water as external standard, 162 MHz, δ ppm): (m, HOPN and CH 2 OPN, 2P), (m, OPOO, 2P); HR-MS (ESI-TOF) (m/z): calcd, ; found, [M - 1] Preparation of polymer-bound mono-, di-, tri-, and tetraphosphitylating reagents (14 17). Polymer-bound bis[diisopropylamino]phosphite (14), Polymer-bound [N,Ndiisopropylphosphoramidite]-O-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite] (15), Polymerbound O-[N,N-diisopropylphosphoramidite]-O-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]- 2-cyanoethylphosphite (16), and Polymer-bound O-[O-[N,N-diisopropylphosphoramidite]-2- cyanoethylphosphite]-o-[(2-cyanoethyl)-n,n-diisopropylphosphoramidite]-2-cyanoethylphosphite (17). The prepared reaction mixtures containing 1, 5, 9 or 12 in THF (~10 mmol) were immediately added to a swelled solution of polymer-bound p-acetoxybenzyl alcohol 13 (3.63 g, 0.69 mmol/g, each) and DIEA (1.75 ml, 10 mmol). The mixtures were shaken for 24 h at 4 C. The resins were collected by filtration, washed with THF (3 35 ml), DCM (3 35 ml), and MeOH (3 35 ml), respectively, and was dried overnight under vacuum to afford 14 (4.19 g, 94%, 0.62 mmol/g), 15 (4.45 g, 94%, 0.53

8 mmol/g), 16 (4.73 g, 95%, 0.50 mmol/g), and 17 (4.97 g, 92%, 0.46 mmol/g). IR (cm -1 ): 14: 1753 S8 (C=O ester), 1027 (P-O-C); 15: 2250 (CN), 1756 (C=O ester), 1028 (P-O-C); 16: 2261 (CN), 1758 (C=O ester), 1029 (P-O-C); 17: 2261 (CN), 1755 (C=O ester), 1028 (P-O-C). 7. Solid-phase synthesis of symmetrical 5,5 -dinucleoside mono-, di-, tri-, and tetraphosphodiesters (31 34a e) using polymer-bound mono-, di-, tri-, and tetraphosphitylating reagents Preparation of polymer-bound 5 -O-5 -O-dinucleoside phosphite triester derivatives 18 21a e. Unprotected nucleosides (a e, 4.0 mmol) and 5-(ethylthio)-1H-tetrazole (260 mg, 2.0 mmol) were added to 14 (638 mg, 0.62 mmol/g), 15 (890 mg, 0.53 mmol/g), 16 (946 mg, 0.50 mmol/g), and 17 (994 mg, 0.46 mmol/g) in anhydrous THF (2 ml) and DMSO (3 ml) in case of thymidine, 3 -azido-3 - deoxythymidine, and inosine or in anhydrous DMSO (5 ml) in case of adenosine and cytidine. The mixtures were shaken for 48 h at room temperature. The resins were collected by filtration and washed with DMSO (3 30 ml), THF (3 30 ml), and MeOH (3 30 ml), respectively, and dried under vacuum to give 18a e ( mg), 19a e ( mg), 20a e ( mg), and 21a e ( mg). IR (cm -1 ): 18a: 3343 (OH), 1759 (C=O ester), 1028 (P-O-C); 18b: 3319 (OH), 1755 (C=O ester), 1029 (P-O-C); 18c: 1767 (C=O ester), 1029 (P-O-C); 18d: 3332 (OH), 1751 (C=O ester), 1028 (P- O-C); 18e: 3344 (OH), 1750 (C=O ester), 1028 (P-O-C); 19a: 3334 (OH), 2255 (CN), 1768 (C=O ester), 1028 (P-O-C); 19b: 3341 (OH), 2253 (CN), 1759 (C=O ester), 1028 (P-O-C); 19c: 2244 (CN), 1766 (C=O ester), 1029 (P-O-C); 19d: 3324 (OH), 2249 (CN), 1760 (C=O ester), 1027 (P-O-C); 19e: 3339 (OH), 2253 (CN), 1756 (C=O ester), 1029 (P-O-C); 20a: 3332 (OH), 2263 (CN), 1759 (C=O ester), 1028 (P-O-C); 20b: 3333 (OH), 2254 (CN), 1757 (C=O ester), 1028 (P-O-C); 20c: 2253 (CN), 1742 (C=O ester), 1028 (P-O-C); 20d: 3345 (OH), 2248 (CN), 1751 (C=O ester), 1028 (P-O-C); 20e: 3342 (OH), 2258 (CN), 1754 (C=O ester), 1029 (P-O-C); 21a: 3336 (OH), 2260 (CN), 1764 (C=O ester), 1027 (P-O- C); 21b: 3336 (OH), 2250 (CN), 1768 (C=O ester), 1028 (P-O-C); 21c: 2260 (CN), 1767 (C=O ester),

9 1029 (P-O-C); 21d: 3333 (OH), 2253 (CN), 1743 (C=O ester), 1028 (P-O-C); 21e: 3326 (OH), 2257 S9 (CN), 1752 (C=O ester), 1028 (P-O-C). Oxidation of polymer-bound 5 -O-5 -O-dinucleoside phosphite triester derivatives 18 21a e to polymer-bound dinucleoside phosphate triester derivatives 22 25a e. tert-butyl hydroperoxide in decane (5-6 M) was added to the swelled resins 18a e ( mg) (tbuooh, 0.32 ml, 1.6 mmol), 19a e ( mg), (tbuooh, 1.0 ml, 5.0 mmol), 20a e ( mg) (tbuooh, 1.5 ml, 7.5 mmol), and 21a e ( mg) (tbuooh, 2.0 ml, 10.0 mmol) in THF (5 ml). After 2.5 h shaking at room temperature, the resins were collected by filtration and washed with THF (3 25 ml) and MeOH (3 25 ml), respectively, and were dried overnight at room temperature under vacuum to give 22a e ( mg), 23a e ( mg), 24a e ( mg), and 25a e ( mg). IR (cm -1 ): 22a: 3326 (OH), 1748 (C=O ester), 1028 (P-O-C); 22b: 3329 (OH), 1765 (C=O ester), 1029 (P-O-C); 22c: 3337 (C=O ester), 1028 (P-O-C); 22d: 3335 (OH), 1757 (C=O ester), 1029 (P-O-C); 22e: 3326 (OH), 1742 (C=O ester), 1029 (P-O-C); 23a: 3330 (OH), 2253 (CN), 1756 (C=O ester), 1030 (P-O-C); 23b: 3349 (OH), 2263 (CN), 1765 (C=O ester), 1029 (P-O-C); 23c: 2258 (CN), 1761 (C=O ester), 1027 (P-O- C); 23d: 3325 (OH), 2266 (CN), 1763 (C=O ester), 1027 (P-O-C); 23e: 3308 (OH), 2256 (CN), 1749 (C=O ester), 1028 (P-O-C); 24a: 3337 (OH), 2260(CN), 1753 (C=O ester), 1028 (P-O-C); 24b: 3345 (OH), 2258 (CN), 1756 (C=O ester), 1028 (P-O-C); 24c: 2249 (CN), 1762 (C=O ester), 1027 (P-O-C); 24d: 3338 (OH), 2252 (CN), 1758 (C=O ester), 1026 (P-O-C); 24e: 3332 (OH), 2253 (CN), 1754 (C=O ester), 1026 (P-O-C); 25a: 3342 (OH), 2263 (CN), 1765 (C=O ester), 1029 (P-O-C); 25b: 3323 (OH), 2250 (CN), 1751 (C=O ester), 1024 (P-O-C); 25c: 2261 (CN), 1757 (C=O ester), 1028 (P-O-C); 25d: 3331 (OH), 2246 (CN), 1750 (C=O ester), 1023 (P-O-C); 25e: 3311 (OH), 2252 (CN), 1754 (C=O ester), 1028 (P-O-C).

10 Preparation of polymer-bound 5 -O-5 -O-dinucleoside phosphate ester derivatives 26 28a e. S10 DBU was added to the swelled resins 23a e ( mg) (DBU, 299 µl, 2.0 mmol), 24a e ( mg) (DBU, 598 µl, 4.0 mmol), and 25a e ( mg) (DBU, 897 µl, 6.0 mmol) in THF (5 ml). After 48 h shaking of the mixture at room temperature, the resins were collected by filtration and washed with THF (3 35 ml) and MeOH (3 35 ml), respectively, and were dried overnight at room temperature under vacuum to give 26a e ( mg), 27a e ( mg), and 28a e ( mg). IR (cm -1 ): 26a: 3332 (O-H), 1749 (C=O ester), 1023 (P-O-C); 26b: 3336 (O-H), 1757 (C=O ester), 1030 (P-O-C); 26c: 1757 (C=O ester), 1026 (P-O-C); 26d: 3342 (O-H), 1753 (C=O ester), 1029 (P- O-C); 26e: 3323 (O-H), 1752 (C=O ester), 1028 (P-O-C); 27a: 3325 (O-H), 1762 (C=O ester), 1029 (P-O- C); 27b: 3327 (O-H), 1761 (C=O ester), 1029 (P-O-C); 27c: 1763 (C=O ester), 1029 (P-O-C); 27d: 3353 (O-H), 1764 (C=O ester), 1028 (P-O-C); 27e: 3334 (O-H), 1743 (C=O ester), 1030 (P-O-C); 28a: 3355 (OH), 1757 (C=O ester), 1029 (P-O-C); 28b: 3352 (OH), 1760 (C=O ester), 1028 (P-O-C); 28c: 2260 (CN), 1762 (C=O ester), 1030 (P-O-C); 28d: 3322 (OH), 1755 (C=O ester), 1029 (P-O-C); 28e: 3349 (OH), 1748 (C=O ester), 1028 (P-O-C). Preparation of dinucleoside 5 -O-5 -O-diphosphate diesters 31 34a e. To the swelled resins 22a e ( mg), 26a e ( mg), 27a e ( mg), and 28a e ( mg) in anhydrous DCM (2 ml) was added DCM/TFA/water/EDT (72.5:23:2.5:2 v/v/v/v, 5 ml). After 30 min shaking of the mixtures at room temperature, the resins were collected by filtration and washed with DCM (10 ml), THF (10 ml), and MeOH (10 ml), respectively. The solvents of filtrate solutions were evaporated at -20 C. The residues were mixed with Amberlite AG-50W-X8 ( mesh, hydrogen form, 1.0 g, 1.7 meq/g) in water:dioxane (75:25 v/v, 5 ml) for 25 min. After filtration, the solvents were removed using lyophilization and the crude products were purified on C 18 Sep-Pak using appropriate solvents. The solvents were evaporated and the residues were dried under vacuum at 20 C to yield 31 34a e. The purity and total isolated yields for 31 34a e are shown in Table S1. The compounds were characterized

11 by 1 H NMR, 13 C NMR, 31 P NMR, high resolution time-of-flight mass spectrometer (ESI-TOF), and S11 quantitative phosphorus elemental analysis. Table S1. Overall Isolated Yields and Purity of Crude Products for 31 34a e. no. overall yield (%) purity of crude products calculated from a b c d e a b c d e a b c d e a b c d e Thymidinyl 5 -thymidinyl phosphate (TpT, 31a). 1 H NMR (DMSO-d 6, 400 MHz, δ ppm): 1.77 (d, J 5-CH3,6 = 1.1 Hz, 5-CH 3, 6H), (m, H-2' and H-2'', 4H), (m, H-5' and H-5'', 4H), (m, H-4', 2H), (m, H-3', 2H), (m, OH, 2H), (m, OH, 2H), 6.20 (t, J 1',2' and J 1',2'' = 8.0 Hz, H-1', 2H), 7.74 (d, J 6,5-CH3 = 1.1 Hz, H-6, 2H), (br s, NH, 2H); 13 C NMR (DMSO-d 6, 100 MHz, δ ppm): 13.0 (5-CH 3 ), 40.2 (C-2'), 62.2 (C-5'), 71.3 (C-3'), 84.6 (C-4'), (C-1'), (C-5), (C-6), (C-2 C=O), (C-4 C=O); 31 P NMR (in DMSO-d 6 and H 3 PO 4 85% in water as external standard, 162 MHz, δ ppm): 4.93 (s, 1P); HR-MS (ESI-TOF) (m/z): calcd, ; found, [M + 1] + ; Anal. calcd, P 5.67%; found, 5.71%.

12 S12 5 -Adenosyl 5 -adenosyl phosphate (ApA, 31b). This compound has been previously synthesized by us H NMR and 13 C NMR were consistent with the previously reported data. 31 P NMR (in DMSO-d 6 and H 3 PO 4 85% in water as external standard, 162 MHz, δ ppm): 4.94 (s, 1P); HR-MS (ESI-TOF) (m/z): calcd, ; found, [M - 1] ; Anal. calcd, P 5.19%, found, 5.24%. 3 -Azido-3 -deoxy-5 -thymidinyl 3 -azido-3 -deoxy-5 -thymidinyl phosphate (AZTpAZT, 31c). 1 H NMR (DMSO-d 6, 400 MHz, δ ppm): (br s, 6H, CH 3 ), (m, H-2', 2H), (m, H-2'', 2H), (m, H-5' and H-5'', 4H), (m, H-4', 2H), (m, H-3', 2H), (br s, OH, 2H), (m, H-1', 2H), (br s, H-6, 2H), (br s, NH, 2H); 13 C NMR (DMSO-d 6, 100 MHz, δ ppm): 13.0 (5-CH 3 ), 37.0 (C-2'), 60.9 (C-3'), 61.6 (C-5'), 84.2 (C- 4'), 84.8 (C-1'), (C-5), (C-6), (C-2 C=O), (C-4 C=O); 31 P NMR (in DMSO-d 6 and H 3 PO 4 85% in water as external standard, 162 MHz, δ ppm): 5.12 (s); HR-MS (ESI-TOF) (m/z): calcd, ; found, [M + H] + ; Anal. calcd, P 5.19%; found, 5.07%. 5 -Cytidyl 5 -cytidyl phosphate (CpC, 31d). 1 H NMR (DMSO-d 6, 400 MHz, δ ppm): (m, H- 5' and H-5'', 4H), (m, H-4', 2H), (m, H-2', H-3', 4H), (br s, 4H, OH), (br s, 2H, OH), (m, H-1', H-5, 4H), (br s, NH 2, 4H), (m, H-6, 2H); 13 C NMR (DMSO-d 6, 100 MHz, δ ppm): 61.8 (C-5'), 70.6 (C-2'), 75.1 (C-3'), 85.3 (C-4'), 90.2 (C- 1'), 95.6 (C-5), (C-6), (C-2 C=O), (C-4 C=O); 31 P NMR (in DMSO-d 6 and H 3 PO 4 85% in water as external standard, 162 MHz, δ ppm): 5.07 (s, 1P); HR-MS (ESI-TOF) (m/z): calcd, ; found, [M + H] + ; Anal. calcd, P 5.65%; found, 5.53%. 5 -Inosinyl 5 -inosinyl phosphate (IpI, 31e). 1 H NMR (DMSO-d 6, 400 MHz, δ ppm): (m, H- 5' and H-5'', 4H), (m, H-4', 2H), (m, H-3', 2H), (m, H-2', 2H),

13 (m, OH, 4H), (m, OH, 2H), (m, H-1', 2H), (br s, H-2, 2H), S13 (br s, H-8, 2H), (br s, OH, 2H); 13 C NMR (DMSO-d 6, 100 MHz, δ ppm): 62.3 (C-5'), 71.3 (C-2'), 75.2 (C-3'), 86.6 (C-4'), 88.6 (C-1'), (C-5), (C-8), (C-4), (C-2), (C- 6); 31 P NMR (in DMSO-d 6 and H 3 PO 4 85% in water as external standard, 162 MHz, δ ppm): 4.76 (s, 1P); HR-MS (ESI-TOF) (m/z): calcd, ; found, [M] + ; Anal. calcd, P 5.18%; found, 5.31%. 5 -Thymidinyl 5 -thymidinyl diphosphate (Tp 2 T, 32a). 1 H NMR (DMSO-d 6, 400 MHz, δ ppm): 1.77 (d, J 5-CH3,6 = 1.1 Hz, 5-CH 3, 6H), (m, H-2' and H-2'', 4H), (m, H-5' and H-5'', 4H), (m, H-4', 2H), (m, H-3', 2H), (m, OH, 2H), (m, OH, 2H), 6.20 (t, J 1',2' and J 1',2'' = 8.0 Hz, H-1', 2H), 7.73 (d, J 6,5-CH3 = 1.1 Hz, H-6, 2H), (br s, NH, 2H); 13 C NMR (DMSO-d 6, 100 MHz, δ ppm): 13.0 (5-CH 3 ), 40.3 (C-2'), 62.1 (C-5'), 71.3 (C-3'), 84.6 (C-4'), 88.1 (C-1'), (C-5), (C-6), (C-2 C=O), (C-4 C=O); 31 P NMR (in DMSO-d 6 and H 3 PO 4 85% in water as external standard, 162 MHz, δ ppm): (br s, 2P); HR-MS (ESI-TOF) (m/z): calcd, ; found, [M + Na] + ; Anal. calcd, P 9.89%; found, 9.57%. 5 -Adenosyl 5 -adenosyl diphosphate (Ap 2 A, 32b). 1 H NMR (DMSO-d 6, 400 MHz, δ ppm): (m, H-5', 2H), (m, H-5'', 2H), (m, H-4', 2H), (m, H-3', 2H), (m, H-2', 2H), (m, OH, 2H), (m, OH, 4H), (m, H-1', 2H), (br s, 6- NH 2, 4H), (br s, H-2, 2H), (br s, H-8, 2H); 13 C NMR (DMSO-d 6, 100 MHz, δ ppm): δ 62.5 (C-5'), 71.4 (C-2'), 74.2 (C-3'), 86.7 (C-4'), 88.7 (C-1'), (C-5), (C-8), (C-4), (C-2), (C-6); 31 P NMR (in DMSO-d 6 and H 3 PO 4 85% in water as external standard, 162 MHz, δ ppm): (br s, 2P); HR-MS (ESI-TOF) (m/z): calcd, ; found, [M + Na + H] + ; Anal. calcd, P 9.16 %; found, 9.27%.

14 S14 3 -Azido-3 -deoxy-5 -thymidinyl 3 -azido-3 -deoxy-5 -thymidinyl diphosphate (AZTp 2 AZT, 32c). 1 H NMR (DMSO-d 6, 400 MHz, δ ppm): (br s, 6H, CH 3 ), (m, H-2', 2H), (m, H-2'', 2H), (m, H-5' and H-5'', 4H), (m, H-4', 2H), (m, H-3', 2H), (br s, OH, 2H), (m, H-1', 2H), (br s, H-6, 2H), (br s, NH, 2H); 13 C NMR (DMSO-d 6, 100 MHz, δ ppm): 13.0 (5-CH 3 ), 37.0 (C-2'), 60.9 (C-3'), 61.6 (C-5'), 84.2 (C- 4'), 84.7 (C-1'), (C-5), (C-6), (C-2 C=O), (C-4 C=O); 31 P NMR (in DMSO-d 6 and H 3 PO 4 85% in water as external standard, 162 MHz, δ ppm): (br s, 2P); HR-MS (ESI-TOF) (m/z): calcd, ; found, [M + Na + H] + ; Anal. calcd, P 9.16%; found, 8.94%. 5 -Cytidyl 5 -cytidyl diphosphate (Cp 2 C, 32d). 1 H NMR (DMSO-d 6, 400 MHz, δ ppm): (m, H-5' and H-5'', 4H), (m, H-4', 2H), (m, H-2', H-3', 4H), (br s, 4H, OH), (br s, 2H, OH), (m, H-1', H-5, 4H), (br s, NH 2, 4H), (m, H-6, 2H); 13 C NMR (DMSO-d 6, 100 MHz, δ ppm): 61.8 (C-5'), 70.6 (C-2'), 75.1 (C-3'), 85.3 (C-4'), 90.1 (C- 1'), 95.5 (C-5), (C-6), (C-2 C=O), (C-4 C=O); 31 P NMR (in DMSO-d 6 and H 3 PO 4 85% in water as external standard, 162 MHz, δ ppm): (br s, 2P); HR-MS (ESI-TOF) (m/z): calcd, ; found, [M + Na + H] + ; Anal. calcd, P 9.86%; found, 10.04%. 5 -Inosinyl 5 -inosinyl diphosphate (Ip 2 I, 32e). 1 H NMR (DMSO-d 6, 400 MHz, δ ppm): (m, H-5' and H-5'', 4H), (m, H-4', 2H), (m, H-3', 2H), (m, H-2', 2H), (m, OH, 4H), (m, OH, 2H), (m, H-1', 2H), (br s, H-2, 2H), (br s, H-8, 2H), (br s, OH, 2H); 13 C NMR (DMSO-d 6, 100 MHz, δ ppm): 62.3 (C- 5'), 71.3 (C-2'), 75.2 (C-3'), 86.6 (C-4'), 88.6 (C-1'), (C-5), (C-8), (C-4), (C-2), (C-6); 31 P NMR (in DMSO-d 6 and H 3 PO 4 85% in water as external standard, 162 MHz, δ ppm): (br s, 2P); HR-MS (ESI-TOF) (m/z): calcd, ; found, [M + Na] + ; Anal. calcd, P 9.13%; found, 9.27%.

15 S15 5 -Thymidinyl 5 -thymidinyl triphosphate (Tp 3 T, 33a). 1 H NMR (DMSO-d 6, 400 MHz, δ ppm): 1.77 (d, J 5-CH3,6 = 1.1 Hz, 5-CH 3, 6H), (m, H-2' and H-2'', 4H), (m, H-5' and H-5'', 4H), (m, H-4', 2H), (m, H-3', 2H), (m, OH, 2H), (m, OH, 2H), 6.16 (t, J 1',2' and J 1',2'' = 8.0 Hz, H-1', 2H), 7.68 (d, J 6,5-CH3 = 1.1 Hz, H-6, 2H), (br s, NH, 2H); 13 C NMR (DMSO-d 6, 100 MHz, δ ppm): 13.0 (5-CH 3 ), 40.2 (C-2'), 62.1 (C-5'), 71.2 (C-3'), 84.5 (C-4'), 88.0 (C-1'), (C-5), (C-6), (C-2 C=O), (C-4 C=O); 31 P NMR (in DMSO-d 6 and H 3 PO 4 85% in water as external standard, 162 MHz, δ ppm): (t, J α-p,β-p = 17.8 Hz, β-p, 1P), (d, J α- P,β-P = 17.8 Hz, α-p, 2P); HR-MS (ESI-TOF) (m/z): calcd, ; found, [M - 1] - ; Anal. calcd, P 13.15%; found, 12.89%. 5 -Adenosyl 5 -adenosyl triphosphate (Ap 3 A, 33b). 1 H NMR (DMSO-d 6, 400 MHz, δ ppm): δ (m, H-5', 2H), (m, H-5'', 2H), (m, H-4', 2H), (m, H-3', 2H), (m, H-2', 2H), (m, OH, 2H), (m, OH, 4H), (m, H-1', 2H), (br s, 6-NH 2, 4H), (br s, H-2, 2H), (br s, H-8, 2H); 13 C NMR (DMSO-d 6, 100 MHz, δ ppm): δ 62.5 (C-5'), 71.5 (C-2'), 74.3 (C-3'), 86.7 (C-4'), 89.8 (C-1'), (C-5), (C- 8), (C-4), (C-2), (C-6); 31 P NMR (in DMSO-d 6 and H 3 PO 4 85% in water as external standard, 162 MHz, δ ppm): (t, J α-p,β-p = 19.4 Hz, β-p, 1P), (d, J α-p,β-p = 19.4 Hz, α-p, 2P);HR-MS (ESI-TOF) (m/z): calcd, ; found, [M] + ; Anal. calcd, P 12.28%; found, 12.09%. 3 -Azido-3 -deoxy-5 -thymidinyl 3 -azido-3 -deoxy-5 -thymidinyl triphosphate (AZTp 3 AZT, 33c). 1 H NMR (DMSO-d 6, 400 MHz, δ ppm): (br s, 6H, CH 3 ), (m, H-2', 2H), (m, H-2'', 2H), (m, H-5' and H-5'', 4H), (m, H-4', 2H), (m, H-3', 2H),

16 S (br s, OH, 2H), (m, H-1', 2H), (br s, H-6, 2H), (br s, NH, 2H); 13 C NMR (DMSO-d 6, 100 MHz, δ ppm): 13.1 (5-CH 3 ), 37.1 (C-2'), 61.0 (C-3'), 61.7 (C-5'), 84.3 (C- 4'), 84.9 (C-1'), (C-5), (C-6), (C-2 C=O), (C-4 C=O); 31 P NMR (in DMSO-d 6 and H 3 PO 4 85% in water as external standard, 162 MHz, δ ppm): (t, J α-p,β-p = 19.4 Hz, β-p, 1P), (d, J α-p,β-p = 19.4 Hz, α-p, 2P); HR-MS (ESI-TOF) (m/z): calcd, ; found, [M] + ; Anal. calcd, P %; found, 12.41%. 5 -Cytidyl 5 -cytidyl triphosphate (Cp 3 C, 33d). 1 H NMR (DMSO-d 6, 400 MHz, δ ppm): (m, H-5' and H-5'', 4H), (m, H-4', 2H), (m, H-2', H-3', 4H), (br s, 4H, OH), (br s, 2H, OH), (m, H-1', H-5, 4H), (br s, NH 2, 4H), (m, H-6, 2H); 13 C NMR (DMSO-d 6, 100 MHz, δ ppm): 61.8 (C-5'), 70.6 (C-2'), 75.1 (C-3'), 85.2 (C-4'), 90.1 (C- 1'), 95.5 (C-5), (C-6), (C-2 C=O), (C-4 C=O); 31 P NMR (in DMSO-d 6 and H 3 PO 4 85% in water as external standard, 162 MHz, δ ppm): (t, J α-p,β-p = 19.4 Hz, β-p, 1P), (d, J α-p,β-p = 19.4 Hz, α-p, 2P); HR-MS (ESI-TOF) (m/z): calcd, ; found, [M + H] + ; Anal. calcd, P %; found, 12.98%. 5 -Inosinyl 5 -inosinyl triphosphate (Ip 3 I, 33e). 1 H NMR (DMSO-d 6, 400 MHz, δ ppm): (m, H-5' and H-5'', 4H), (m, H-4', 2H), (m, H-3', 2H), (m, H-2', 2H), (m, OH, 4H), (m, OH, 2H), (m, H-1', 2H), (br s, H-2, 2H), (br s, H-8, 2H), (br s, OH, 2H); 13 C NMR (DMSO-d 6, 100 MHz, δ ppm): 62.3 (C- 5'), 71.3 (C-2'), 75.2 (C-3'), 86.7 (C-4'), 88.6 (C-1'), (C-5), (C-8), (C-4), (C-2), (C-6); 31 P NMR (in DMSO-d 6 and H 3 PO 4 85% in water as external standard, 162 MHz, δ ppm): (t, J α-p,β-p = 17.8 Hz, β-p, 1P), (d, J α-p,β-p = 17.8 Hz, α-p, 2P); HR-MS (ESI-TOF) (m/z): calcd, ; found, [M - 1] - ; Anal. calcd, P 12.25%; found, 12.27%.

17 S17 5 -Thymidinyl 5 -thymidinyl tetraphosphate (Tp 4 T, 34a). 1 H NMR (DMSO-d 6, 400 MHz, δ ppm): 1.77 (d, J 5-CH3,6 = 1.1 Hz, 5-CH 3, 6H), (m, H-2' and H-2'', 4H), (m, H-5' and H-5'', 4H), (m, H-4', 2H), (m, H-3', 2H), (m, OH, 2H), (m, OH, 2H), 6.18 (t, J 1',2' and J 1',2'' = 8.0 Hz, H-1', 2H), 7.70 (d, J 6,5-CH3 = 1.1 Hz, H-6, 2H), (br s, NH, 2H); 13 C NMR (DMSO-d 6, 100 MHz, δ ppm): 13.0 (5-CH 3 ), 40.3 (C-2'), 62.1 (C-5'), 71.2 (C-3'), 84.5 (C-4'), 88.1 (C-1'), (C-5), (C-6), (C-2 C=O), (C-4 C=O); 31 P NMR (in DMSO-d 6 and H 3 PO 4 85% in water as external standard, 162 MHz, δ ppm): (d, J α-p,β-p = 18.6 Hz, β-p, 2P), (d, J α-p,β-p = 18.6 Hz, α-p, 2P); HR-MS (ESI-TOF) (m/z): calcd, ; found, [M - 1] - ; Anal. calcd, P 15.76%; found, 15.59%. 5 -Adenosyl 5 -adenosyl tetraphosphate (Ap 4 A, 34b). 1 H NMR (DMSO-d 6, 400 MHz, δ ppm): δ (m, H-5', 2H), (m, H-5'', 2H), (m, H-4', 2H), (m, H-3', 2H), (m, H-2', 2H), (m, OH, 2H), (m, OH, 4H), (m, H-1', 2H), (br s, 6-NH 2, 4H), (br s, H-2, 2H), (br s, H-8, 2H); 13 C NMR (DMSO-d 6, 100 MHz, δ ppm): δ 62.5 (C-5'), 71.5 (C-2'), 74.3 (C-3'), 86.7 (C-4'), 88.8 (C-1'), (C-5), (C- 8), (C-4), (C-2), (C-6); 31 P NMR (in DMSO-d 6 and H 3 PO 4 85% in water as external standard, 162 MHz, δ ppm: (d, J α-p,β-p = 18.6 Hz, β-p, 2P), (d, J α-p,β-p = 18.6 Hz, α-p, 2P); HR-MS (ESI-TOF) (m/z): calcd, ; found, [M] + ; Anal. calcd, P 14.81%; found, 14.67%. 3 -Azido-3 -deoxy-5 -thymidinyl 3 -azido-3 -deoxy-5 -thymidinyl tetraphosphate (AZTp 4 AZT, 34c). 1 H NMR (DMSO-d 6, 400 MHz, δ ppm): (br s, 6H, CH 3 ), (m, H-2', 2H), (m, H-2'', 2H), (m, H-5' and H-5'', 4H), (m, H-4', 2H), (m, H-3', 2H), (br s, OH, 2H), (m, H-1', 2H), (br s, H-6, 2H), (br s, NH,

18 S18 2H); 13 C NMR (DMSO-d 6, 100 MHz, δ ppm): 13.0 (5-CH 3 ), 37.0 (C-2'), 60.9 (C-3'), 61.6 (C-5'), 84.1 (C-4'), 84.7 (C-1'), (C-5), (C-6), (C-2 C=O), (C-4 C=O); 31 P NMR (in DMSO-d 6 and H 3 PO 4 85% in water as external standard, 162 MHz, δ ppm): (d, J α-p,β-p = 17.8 Hz, β-p, 2P), (d, J α-p,β-p = 17.8 Hz, α-p, 2P); HR-MS (ESI-TOF) (m/z): calcd, ; found, [M] + ; Anal. calcd, P 14.81%; found, 15.12%. 5 -Cytidyl 5 -cytidyl tetraphosphate (Cp 4 C, 34d). 1 H NMR (DMSO-d 6, 400 MHz, δ ppm): (m, H-5' and H-5'', 4H), (m, H-4', 2H), (m, H-2', H-3', 4H), (br s, 4H, OH), (br s, 2H, OH), (m, H-1', H-5, 4H), (br s, NH 2, 4H), (m, H-6, 2H); 13 C NMR (DMSO-d 6, 100 MHz, δ ppm): 61.8 (C-5'), 70.6 (C-2'), 75.1 (C-3'), 85.3 (C-4'), 90.2 (C-1'), 95.6 (C-5), (C-6), (C-2 C=O), (C-4 C=O); 31 P NMR (in DMSO-d 6 and H 3 PO 4 85% in water as external standard, 162 MHz, δ ppm): (d, J α-p,β-p = 19.4 Hz, β-p, 2P), (d, J α- P,β-P = 19.4 Hz, α-p, 2P); HR-MS (ESI-TOF) (m/z): calcd, ; found, [M - 1] - ; Anal. calcd, P %; found, 15.98%. 5 -Inosinyl 5 -inosinyl tetraphosphate (Ip 4 I, 34e). 1 H NMR (DMSO-d 6, 400 MHz, δ ppm): (m, H-5' and H-5'', 4H), (m, H-4', 2H), (m, H-3', 2H), (m, H-2', 2H), (m, OH, 4H), (m, OH, 2H), (m, H-1', 2H), (br s, H-2, 2H), (br s, H-8, 2H), (br s, OH, 2H); 13 C NMR (DMSO-d 6, 100 MHz, δ ppm): 62.2 (C- 5'), 71.3 (C-2'), 75.1 (C-3'), 86.6 (C-4'), 88.5 (C-1'), (C-5), (C-8), (C-4), (C-2), (C-6); 31 P NMR (in DMSO-d 6 and H 3 PO 4 85% in water as external standard, 162 MHz, δ ppm): (d, J α-p,β-p = 178 Hz, α-p, 2P), (d, J α-p,β-p = 17.8 Hz, β-p, 2P); HR-MS (ESI-TOF) (m/z): calcd, ; found, [M + 1] + ; Anal. calcd, P 14.78%; found, 14.57%.

19 8. 1 H NMR, 13 C NMR, and 31 P NMR spectra of hydroxyphosphoramidite form of mono-, di-, S19 tri-, and tetraphosphitylating reagents (1, 5, 9, 12 ) and symmetrical 5,5 -dinucleoside mono-, di-, tri-, and tetraphosphodiesters (31-34a e).

20 S20

21 S21

22 S22

23 S23

24 S24

25 S25

26 S26

27 S27

28 S28

29 S29

30 S30

31 S31

32 S32

33 S33

34 S34

35 S35

36 4 S36

37 S37

38 S38

39 S39

40 S40

41 S41

42 S42

43 S43

44 S44

45 S45

46 S46

47 S47

48 S48

49 S49

50 S50

51 S51

52 S52

53 S53

54 S54

55 9. Analytical HPLC Profile of Final Compounds S55 The purity of final products was confirmed by analytical HPLC. The analytical HPLC analysis was performed on a Beckman Ultrasphere TM analytical C 18 reversed-phase column ( cm) with a Hitachi Elite LaChrom TM series 2000 analytical HPLC instrument using a gradient system of H 2 O (15 mm ammonium acetate) and methanol at 260 nm. The analysis was performed with a gradient of 25-35% CH 3 OH over 10 min and a flow rate of ml/min. The HPLC system consisted of a L-2130 low pressure gradient pump and high sensitivity diode array detector ( nm). Table S2. HPLC Gradient System. Time (min) Water (15 mm Methanol Flow Rate (ml/min) ammonium acetate) (%) (%) Table S3. The retention times (min.) of the final products in analytical HPLC. Compound No. Retention Time (min) Compound No. 31a a b b c c d d e e a a b b c c d d e e 6.25 Retention Time (min)

56 DAD-CH3 260 nm 31a.dat 31a Retention time = 5.87 min S mau Minutes DAD-CH3 260 nm 31b.dat 31b Retention time = 6.69 min mau Minutes

57 DAD-CH3 260 nm 31c.dat 31c Retention time = 6.38 min S mau Minutes DAD-CH3 260 nm 31d.dat 31d Retention time = 7.18 min mau Minutes

58 DAD-CH3 260 nm 31e.dat 31e Retention time = 5.97 min S mau Minutes DAD-CH3 260 nm 32a.dat 32a Retention time = 5.67 min mau Minutes

59 DAD-CH3 260 nm 32b.dat 32b Retention time = 6.64 min S mau Minutes DAD-CH3 260 nm 32c 32c.dat Retention time = 6.43 min mau Minutes

60 DAD-CH3 260 nm 32d.dat 32d Retention time = 7.05 min S mau Minutes DAD-CH3 260 nm 32e.dat 32e Retention time = 5.81 min mau Minutes

61 DAD-CH3 260 nm 33a.dat 33a Retention time = 5.61 min S mau Minutes DAD-CH3 260 nm 33b.dat 33b Retention time = 7.15 min mau Minutes

62 DAD-CH3 260 nm 33c.dat 33c Retention time = 6.41 min S mau Minutes DAD-CH3 260 nm 33d.dat 33d Retention time = 6.37 min mau Minutes

63 DAD-CH3 260 nm 33e.dat 33e Retention time = 6.34 min S mau Minutes DAD-CH3 260 nm 34a.dat 34a Retention time = 5.69 min mau Minutes

64 DAD-CH3 260 nm 34b.dat 34b Retention time = 7.12 min S mau Minutes DAD-CH3 260 nm 34c 34c.dat Retention time = 6.38 min mau Minutes

65 DAD-CH3 260 nm 34d.dat 34d Retention time = 7.03 min S mau Minutes DAD-CH3 260 nm 34e.dat 34e Retention time = 6.25 min mau Minutes