Supporting Information Available

(1)

Supporting Information Available

Innovative 2’-O-Imino-2-Propanoate Protecting Group for Effective Solid-Phase Synthesis

and 2’-O-Deprotection of RNA Sequences

Mayumi Takahashi, Andrzej Grajkowski, Brian M. Cawrse and Serge L. Beaucage

*

Laboratory of Biological Chemistry, Division of Biotechnology Review and Research IV, Center for Drug Evaluation and Research, Food and Drug Administration, 10903 New Hampshire Avenue, Silver Spring, Maryland 20993

Table of Contents

Materials and Methods………... S04 Figure S1A. 300 MHz 1H-NMR spectrum of ribonucleoside 2 in DMSO-d6………... S06

Figure S1B. 75 MHz 13C {1H} NMR spectrum of ribonucleoside 2 in DMSO-d6………... S06

Figure S2A.300 MHz 1H-NMR spectrum of ribonucleoside 4 in DMSO-d6………... S07

Figure S5. 121 MHz 31P {1H} NMR spectrum of ribonucleoside phosphoramidite 8 in CDCl3……….. S10

Figure S6A.300 MHz 1H-NMR spectrum of ribonucleoside 10 in DMSO-d6………. S11

Figure S6B. 75 MHz 13C {1H} NMR spectrum of ribonucleoside 10 in DMSO-d6………. S11

Figure S9B. 75 MHz 13_{C {1H} NMR spectrum of ribonucleoside 15 in DMSO-d}

6………. S14

(2)

6………... S20

Figure S16. 121 MHz 31P {1H} NMR spectrum of ribonucleoside phosphoramidite 23 in CDCl3………….. S21

6………... S22

Figure S19A.300 MHz 1H-NMR spectrum of ribonucleoside 27in DMSO-d6……… S24

6………... S24

Figure S22. 121 MHz 31P {1H} NMR spectrum of ribonucleoside phosphoramidite 30 in CDCl3…………...S27

Figure S23. APCI-HRMS spectrum of ribonucleoside 2………...………... S28 Figure S24. APCI-HRMS spectrum of ribonucleoside 4……….. S28 Figure S25. APCI-HRMS spectrum of ribonucleoside 6………...………... S29 Figure S26. APCI-HRMS spectrum of ribonucleoside 7……….. S29 Figure S27.APCI-HRMS spectrum of ribonucleoside phosphoramidite 8……….. S30 Figure S28. APCI-HRMS spectrum of ribonucleoside 10……… S30 Figure S29. APCI-HRMS spectrum of ribonucleoside 12………...………. S31 Figure S30. APCI-HRMS spectrum of ribonucleoside 13……… S31 Figure S31. APCI-HRMS spectrum of ribonucleoside 15………...………. S32 Figure S32. APCI-HRMS spectrum of ribonucleoside 16……… S32 Figure S33. APCI-HRMS spectrum of ribonucleoside phosphoramidite 17………. S33 Figure S34. APCI-HRMS spectrum of ribonucleoside 18………...………. S33 Figure S35. APCI-HRMS spectrum of ribonucleoside 19……… S34

(3)

Figure S36. APCI-HRMS spectrum of ribonucleoside 21………...………. S34 Figure S37. APCI-HRMS spectrum of ribonucleoside 22……… S35 Figure S38. APCI-HRMS spectrum of ribonucleoside phosphoramidite 23………. S35 Figure S39. APCI-HRMS spectrum of ribonucleoside 25………...………. S36 Figure S40. APCI-HRMS spectrum of ribonucleoside 26……… S36 Figure S41. APCI-HRMS spectrum of ribonucleoside 27………...………. S37 Figure S42. APCI-HRMS spectrum of ribonucleoside 28……… S37 Figure S43. APCI-HRMS spectrum of ribonucleoside 29……… S38 Figure S44. APCI-HRMS spectrum of ribonucleoside phosphoramidite 30………. S38 Figure S45. RP-HPLC profiles demonstrating the sequential conversion of

the ribonucleoside 6 to uridine………... S39 Figure S46. RP-HPLC profiles demonstrating the sequential conversion of

the ribonucleoside 15 to adenosine………. S39 Figure S47. RP-HPLC profiles demonstrating the sequential conversion of

the ribonucleoside 21 to cytidine……….... S40 Figure S48. RP-HPLC profile of fully deprotected, unpurified r(AUGAGUAGCGAACGUGAAGT)

synthesized from ribonucleoside phosphoramidites 8, 17, 23, and 30 ……….. S41 Figure S49. Expanded RP-HPLC profile of fully deprotected, unpurified

r(AUGAGUAGCGAACGUGAAGT) synthesized from ribonucleoside phosphoramidites

8, 17, 23, and 30 ………. S41 Figure S50. RP-HPLC profile of fully deprotected, unpurified r(AUGAGUAGCGAACGUGAAGT)

synthesized from commercial 2’-O-TBDMS ribonucleoside phosphoramidites………... S42 Figure S51. Expanded RP-HPLC profile of fully deprotected, unpurified

r(AUGAGUAGCGAACGUGAAGT) synthesized from commercial

2’-O-TBDMS ribonucleoside phosphoramidites………... S42 Figure S52. Overlaid RP-HPLC profiles of fully deprotected, unpurified

r(AUGAGUAGCGAACGUGAAGT) synthesized from ribonucleoside phosphoramidites 8, 17, 23, and 30 (blue profile) or from commercial 2’-O-TBDMS ribonucleoside

phosphoramidites (red profile)………... S43 Figure S53. RP-HPLC profile of fully deprotected, unpurified

(4)

Figure S54. Expanded RP-HPLC profile of fully deprotected, unpurified

r(UAUCCGUAGCUAACGUCAGT) synthesized from ribonucleoside phosphoramidites

8, 17, 23, and 30………. S44 Figure S55.RP-HPLC profile of fully deprotected, unpurified

r(UAUCCGUAGCUAACGUCAGT) synthesized from commercial 2’-O-TBDMS

ribonucleoside phosphoramidites………... S45 Figure S56. Expanded RP-HPLC profile of fully deprotected, unpurified

r(UAUCCGUAGCUAACGUCAGT) synthesized from commercial

2’-O-TBDMS ribonucleoside phosphoramidites……… S45 Figure S57. Overlaid RP-HPLC profiles of fully deprotected, unpurified

r(UAUCCGUAGCUAACGUCAGT) synthesized from ribonucleoside phosphoramidites 8, 17, 23, and 30 (blue profile) or from commercial 2’-O-TBDMS ribonucleoside

phosphoramidites (red profile)………... S46 Figure S58. MALDI-TOF MS spectrum of r(AUGAGUAGCGAACGUGAAGT)

synthesized from ribonucleoside phosphoramidites 8, 17, 23, and 30………... S47 Figure S59. MALDI-TOF MS spectrum of r(AUGAGUAGCGAACGUGAAGT)

synthesized from commercial 2’-O-TBDMS ribonucleoside phosphoramidites.……….. S48 Figure S60. MALDI-TOF mass spectrum of r(UAUCCGUAGCUAACGUCAGT)

synthesized from ribonucleoside phosphoramidites 8, 17, 23, and 30………... S49 Figure S61. MALDI-TOF mass spectrum of r(UAUCCGUAGCUAACGUCAGT)

synthesized from commercial 2’-O-TBDMS ribonucleoside phosphoramidites………... S50

Materials and Methods. Specialty and common chemical reagents and solvents including 1-

β-D-arabinofuranosyluracil, 1-β-D-arabinofuranosyladenine, 2,6-diamino-9-β-D-arabinofuranosyl-purine, 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane, 2,4,6-triisopropylbenzenesulfonyl chloride, trifluoromethanesulfonic anhydride, 4-dimethylaminopyridine, N-hydroxyphthalimide, ethyl pyruvate, chlorotrimethylsilane,

phenoxyacetic anhydride, methylhydrazine, ammonium fluoride, butylammonium fluoride, tetra-n-butylammonium chloride, sodium nitrite, 4,4’-dimethoxytrityl chloride, 2-cyanoethyl

N,N-diisopropylchlorophosphoramidite, N,N-diisopropylethylamine, triethylamine, anhydrous pyridine,

1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), anhydrous dichloromethane, anhydrous acetonitrile, anhydrous DMF, anhydrous DMSO, methanol, acetic acid, tetrahydrofuran, concentrated ammonium hydroxide, and deuterated solvents (CDCl3, DMSO-d6) were purchased from reputable commercial sources and used as received.

(5)

Reagents for solid-phase synthesis of RNA sequences including the following: (i) a succinyl long chain alkylamine controlled-pore glass (CPG) support functionalized with 2’-deoxythymidine as the leader

nucleoside; (ii) ribonucleoside 2’-O-(tert-butyldimethylsilyl) phosphoramidite monomers; (iii) 5’-deblocking solution (3% TCA in CH2Cl2); (iv) activator solution (0.25 M 5-ethylthio-1H-tetrazole in MeCN); (v) cap A

solution (Ac2O/pyridine/THF); (vi) cap B solution (10% 1-methylimidazole in THF) and; (vii) oxidizing

solution (0.02 M iodine in THF/pyridine/water) were purchased from commercial sources and used as received. Reagents for deprotection, characterization and processing of synthetic nucleic acid sequences include: aqueous concentrated ammonia, triethylamine trihydrofluoride, 1 M tetra-n-butylammonium fluoride in THF,

triethylammonium acetate buffer (pH 7.0), HPLC grade water and RNase free water were commercially available and used without further purification.

Flash chromatography purifications were performed on glass column (3.0 cm I.D.) packed with silica gel 60 (EMD, 230-400 mesh) or neutral silica gel 60 (Nacalai) when deemed necessary. Analytical thin-layer

chromatography (TLC) analyses were conducted on 2.5 cm × 7.5 cm glass plates coated with a 0.25 mm thick layer of silica gel 60 F254 (EMD). Analytical RP-HPLC analyses were performed using an instrument equipped

with a UV diode array detector operating at 254 nm and a 5 μm Supelcosil LC-18S column (25 cm × 4.6 mm) according to the following conditions: starting from 0.1 M triethylammonium acetate pH 7.0, a linear gradient of 2.5% MeCN/min was pumped at a flow rate of 1 mL/min for 40 min. 2 M Triethylammonium acetate buffer pH 7.0 was purchased from ThermoFisher Scientific and diluted to 0.1 M with HPLC grade water prior to use. All NMR experiments were performed using a spectrometer operating at 300.13, 75.47 and 121.5 MHz for one-dimensional 1_H,1_H-decoupled13_{C and}1_H-decoupled31_{P, respectively. Samples were maintained at a}

temperature of 298 °K. All spectra were recorded in deuterated solvents or as indicated and chemical shifts δ are reported in parts per million (ppm) relative to appropriate internal references. Elemental composition of new compounds was determined, under contract, by high resolution mass spectrometry (HRMS) using a Bruker 12T solariX FT-ICR mass spectrometer operating under and APCI positive ionization mode.

RP-HPLC analyses of synthetic nucleic acid sequences were performed using an Agilent ion-pair reversed-phase AdvanceBio Oligonuleotide column under the following chromatographic conditions: from 0.1 M triethylammonium acetate (pH 7.0), a linear gradient of 0.66% MeCN/min was pumped at a flow rate of 0.8 mL/min for 30 min. Mass determination of each nucleic acid sequence released from each CPG support was performed using a 10 μM solution of each nucleic acid sequence in water or RNase free-water. MALDI-TOF mass spectrometry analyses were carried out under positive polarity and delayed extraction reflector mode; 3-hydroxypicolinic acid [50 mg/mL in MeCN/H O (1:1 v/v)] was used as a calibration matrix and ammonium

(6)

Figure S1A. 300 MHz 1_{H-NMR spectrum of ribonucleoside 2 in DMSO-d} 6

Figure S1B. 75 MHz 13_{C-NMR spectrum of ribonucleoside 2 in DMSO-d} 6

(7)