A drop of HPLC eluent was manually collected, concentrated in vacuo and ..... vortexed on a Vortex Genie-2 apparatus (Scientific Industries) for 1 minute.
Supporting Information
Efficient Solid-Phase Chemical Synthesis of 5'-Triphosphates of DNA, RNA, and their Analogs
Ivan Zlatev,a,b Thomas Lavergne,a Françoise Debart,a* Jean-Jacques Vasseur,a Muthiah Manoharan,b* and François Morvana*
a
Institut des Biomolécules Max Mousseron (IBMM), UMR 5247 CNRS - Université
Montpellier 1 - Université Montpellier 2, Place Eugène Bataillon, CC1704, 34095 Montpellier Cedex 5, France b
Drug Discovery, Alnylam Pharmaceuticals, 300 Third Street, Cambridge, MA, 02142, USA
Table of Contents:
page
I.
General Analytical Procedures
S2
II.
General Procedures for Oligonucleotide Synthesis
S4
III.
General Procedure for 5’-Phosphitylation using Diphenyl Phosphite
S5
IV.
General Procedure for Amidative Oxidation
S6
V.
General Procedure for Phosphorylation using Tributylammonium
S7
Pyrophosphate VI.
Processing of DNA TPs
S8
VII.
Processing of 2’-O-TBDMS and 2’-O-Me protected RNA TPs
S9
VIII. Processing of 2’-O-PivOM protected RNA TPs
S12
IX.
Table S1: Summary for the Prepared Oligonucleotide TPs
S13
X.
Supplementary Experimental Figures
S14
S1
I. General Analytical Procedures
1. Analytical HPLC Method A: Analytical ion-exchange HPLC was performed using a Nucleogel SAX 1000-8 (4.6 mm) anion-exchange column from Macherey-Nagel, installed on a Waters apparatus, equipped with an UV detector at the wavelength of 260 nm and the Millenium software. Compounds were eluted using a linear gradient of 0 to 0.5 M NaCl in a 20 mM sodium phosphate buffer (pH 7.2) in 40 minutes. Flow rate was 1 mL.min-1.
Method B: Analytical HPLC was performed using a BioLC DNAPac PA-100 column (4 × 205 mm) anion-exchange column from Dionex, installed on a Waters apparatus, equipped with an UV detector at the wavelength of 260 nm and the Empower software. Compounds were eluted using a linear gradient of 0 to 0.5 M NaClO4 in a 25 mM TRIZMA HCl buffer (pH 8.0) and 10% acetonitrile in 40 minutes.
Method C: Analytical HPLC was performed using identical elution conditions to those of Method B except that the column was heated to 50 oC.
2. MALDI/TOF mass spectra MALDI/TOF mass spectra were recorded on a Perspective Biosystems Voyager DE spectrometer using a 10:1 (m/m) mixture of 2,4,6-trihydroxyacetophenone/ammonium citrate as a saturated solution in acetonitrile/water– (1:1, v/v) for the matrix. Analytic samples were mixed with the matrix in a 1:5 (v/v) ratio, crystallized on a 100-well stainless steel plate and analyzed. MALDI conditions were: accelerating voltage 24000 V; guide wire 0.05% of the accelerating voltage; grid voltage 94% of the accelerating voltage, delay extraction time 550 ns.
3. Manual analytical LC/MALDI-MS A drop of HPLC eluent was manually collected, concentrated in vacuo and desalted by the addition of several beads of a DOWEX-50W-X8-ammonium resin (Aldrich). After shaking, the supernatant was decanted and mixed with matrix in a 1:1 ratio. The sample was then crystallized on a 100-well stainless steel plate and analyzed by MALDI/TOF MS as described above. S2
4. NMR analysis Method A: 31P NMR spectra were recorded on a Brüker DRX-300 spectrometer at 121 MHz on the proton-decoupled mode. The relaxation delay (d1) parameter was set on 2 sec (default). Samples were dissolved in 0.6 mL of 50% D2O (Eurisotope) and 50% aqueous 10 mM EDTA (Sigma, 3.4 mg/mL). Chemical shifts are given in ppm referenced to external 80% H3PO4; coupling constants are given in Hertz. Method B:
31
P NMR spectra were recorded on a Varian spectrometer at 161 MHz on the
proton-decoupled mode with a relaxation delay (d1) of 0.3 sec. Samples were dissolved in 0.6 mL of 50% D2O (Cambridge Isotope Labs) and 50% aqueous 10 mM EDTA (Sigma, 3.4 mg/mL) in 40 mM TRIZMA-HCl (Sigma, 40 μL). Chemical shifts are given in ppm referenced to external 80% H3PO4; coupling constants are given in Hertz. 5. UV quantitation of ONs UV spectra were recorded on a Varian Cary 300 Bio UV/Visible spectrometer at the wavelength of 260 nm. Typically, the entire yield of an ON synthesis reaction was dissolved in 1000 μL of sterile water. The UV spectrum of 10 µL of this diluted to 1000 μL was recorded. The number of optical density units at 260 nm (OD260) was calculated as the product of the 260 nm absorbance value and the dilution factor (100). Yields were calculated as the ratio of the OD260 value and the calculated OD260 value (product of the synthesis scale in μmol and the calculated epsilon value for each oligonucleotide).
S3
II. General Procedures for Oligonucleotide Synthesis
1. DNA synthesis DNA oligonucleotides were synthesized on an ABI 381A synthesizer from Applied Biosystems. CPG support bearing LCAA succinyl linked dT nucleoside was purchased from Glen Research. 5’-O-DMTr-[dT, dC(Ac), dA(Pac), dG(iPrPac)]-3’-O-(O-cyanoethyl-N,N-diisopropyl) phosphoramidites were purchased from ChemGenes. Phosphoramidites were vacuum dried then dissolved in dry acetonitrile (Glen Research) at 75 mM. Benzylmercaptotetrazole solution (BMT, 0.25 M) was purchased from Fluka. Acetonitrile, dichloroacetic acid (DCA), capping solution (Pac2O) and oxidizing solution (I2/H2O) were purchased from Glen Research. Synthesis was performed on a 1 µmol scale in a 1-µmol Twist oligonucleotide synthesis column (Glen Research) and using the standard 1 μmol DNA “5’ trityl off” synthesis cycle provided by the ABI. After completion of the synthesis cycle, the column was removed from the synthesizer and dried under argon.
2. 2’-O-TBDMS RNA synthesis Synthesis was performed on an ABI 394A synthesizer. CPG support bearing the LCAA succinyl linked corresponding 2’-O-Ac-A(Pac) nucleoside was purchased from ChemGenes. CPG supported dT nucleoside was purchased from Glen Research. 5’-O-DMTr-2’-OTBDMS-[U,
C(Ac),
A(Pac),
G(iPrPac)]-3’-O-(O-cyanoethyl-N,N-di-isopropyl)
phosphoramidites were purchased from ChemGenes. Phosphoramidites were vacuum dried then dissolved in dry acetonitrile (Glen Research) at 200 mM. Ethylthiotetrazole solution (ETT, 0.25 M) was purchased from Glen Research. Acetonitrile, dichloroacetic acid (DCA), capping solution (Pac2O) and oxidizing solution (I2/H2O) were purchased from Glen Research. Sulfurization was performed using a saturated solution of sulfurizing reagent II (DDTT, Glen) in pyridine prepared in house. Synthesis was performed on a 10 µmol scale in a 10-µmol Twist oligonucleotide synthesis column (Glen Research) and using the standard 10-µmol RNA “5’ trityl off” synthesis cycle, provided by ABI. After completion of the synthesis cycle, the column was removed from the synthesizer and dried under argon.
3. 2’-O-PivOM RNA synthesis Synthesis was performed on an ABI 381A synthesizer. CPG supports bearing LCAA succinyl linked 2’-O-Ac-U and 2’-O-Ac-C(Ac) nucleosides were purchased from Glen Research. 5’-ODMTr-2’-O-PivOM-[U, C(Ac), A(Pac), G(iPrPac)]-3’-O-(O-cyanoethyl-N,N-di-isopropyl) S4
phosphoramidites were prepared
according to the reported procedure. 1 For each
incorporation, 20 µmol of phosphoramidite was used. Phosphoramidites were vacuum dried then dissolved in acetonitrile (Glen Research) at 100 mM. Benzylmercaptotetrazole (BMT) was purchased from Chem Genes. Acetonitrile, dichloroacetic acid (DCA), capping solution (Pac2O), and oxidizing solution (I2/H2O) were purchased from Glen Research. Synthesis was performed on a 1 µmol scale in a 1-µmol Twist oligonucleotide synthesis column (Glen Research) and using the standard “5’ trityl off” synthesis cycle provided by ABI. After completion of the synthesis cycle, the column was removed from the synthesizer and dried under argon.
III. General Procedure for 5’-Phosphitylation using Diphenyl Phosphite 1. 0.25 to 4 μmol synthesis scale 0.25 to 4 μmol of the appropriate solid-supported 5’-OH oligonucleotides (see section II) was placed in a dry 1-μmol Twist oligonucleotide synthesis column (Glen Research). The column was closed and flushed with argon. Two mL of a 1 M solution of diphenyl phosphite (Aldrich) in anhydrous pyridine (Aldrich)were pushed manually through the column; using two syringes, the solution was pushed back and forth for 5 minutes at room temperature and then left to react for 30 minutes. The solution was removed from the column, the support was washed with anhydrous acetonitrile and then dried under a stream of argon. Two mL of 100 mM aqueous TEAB (pH = 8.0, Aldrich) was pushed back and forth through the column for 5 min using two syringes and was then left to react for 2 hours. The solution was removed and the column was washed with anhydrous acetonitrile and reverse-flushed with argon. It was placed under vacuum over P2O5 for 24 hours, and was then stored at -20 oC. The conversion rate of the oligonucleotide to its 5’ H-phosphonate monoester, using this method, is above 95%. The reaction was monitored by HPLC and MALDI/TOF MS after deprotection of an aliquot of the beads (using ammonia or/and TBAF) . 2. 10 μmol synthesis scale The appropriate solid-supported 5’OH oligonucleotide (10 μmol) was placed in a dry 10 μmol Twist oligonucleotide synthesis column (Glen Research). The column was closed and flushed
1
Lavergne, T.; Bertrand, J. R.; Vasseur, J. J.; Debart, F., Chem. Eur. J., 2008, 14, (30), 9135-9138.
S5
with argon. Ten mL of a 1 M solution of diphenyl phosphite (Aldrich) in anhydrous pyridine (Aldrich) were pushed back and forth through the column using two syringes for 5 minutes at room temperature and then left to react for 30 minutes. The solution was removed from the column and the support was washed with anhydrous acetonitrile and dried under stream of argon. Ten mL of 100 mM aqueous TEAB (pH = 8.0, Aldrich) was then pushed back and forth through the column for 5 minutes and left to react for 2 hours. The solution was removed and the support was washed with anhydrous acetonitrile and reverse-flushed with argon. It was then placed under vacuum over P2O5 for 24 hours and stored at -20 oC.
IV. General Procedure for Amidative Oxidation 1. Oxidation at the 0.25 to 4 μmol synthesis scale The appropriate solid-supported 5’-H-phosphonate oligonucleotide (0.25 to 4 μmol) was placed in a dry 1-μmol Twist oligonucleotide synthesis column (Glen Research). Activated 4Å molecular sieves (3 to 5 beads) were added to the column. The column was closed and flushed with argon. The oxidation solution was then prepared as follows: 150 mg (2 mmol) of imidazole (Aldrich) were coevaporated twice with anhydrous acetonitrile and then dried under vaccum over P2O5. The residue was redissolved in anhydrous acetonitrile (Glen Research, 0.8 mL), anhydrous CCl4 (Aldrich, 0.8 mL), anhydrous triethylamine (Sigma, 0.1 mL), and N,Obis-trimethylsilyl acetamide (Aldrich, 0.4 mL). The resulting solution was dried over activated 4 Å molecular sieves for 2 min, and then degassed with dry argon for 30 seconds. The solution was pushed back and forth through the column containing the solid-supported 5’-H-phosphonate oligonucleotides using two syringes and then allowed to react for 5 hours at room temperature. The solution was removed from the column and the support was washed twice with methanol, then reverse-flushed with argon. 2. Oxidation at the 10-μmol synthesis scale The appropriate solid-supported 5’-H-phosphonate oligonucleotide (10 μmol) was placed in a dry 10-μmol Twist oligonucleotide synthesis column (Glen Research). Ten to 20 beads of activated 4-Å molecular sieves were added to the column. The column was closed and flushed with argon. The oxidation solution was then prepared as follows: 700 mg (10 mmol) of imidazole (Aldrich) were coevaporated twice with anhydrous acetonitrile and then dried under vaccum over P2O5. The residue was redissolved in anhydrous acetonitrile (Glen, 4 mL),
S6
anhydrous CCl4 (Aldrich, 4 mL), anhydrous triethylamine (Sigma, 0.5 mL), and N,O-bistrimethylsilyl acetamide (Aldrich, 2.5 mL). The resulting solution was dried over activated 4Å molecular sieves for 2 min, and then degassed with dry argon for 30 seconds. The solution was pushed back and forth through the column containing the solid-supported 5’-Hphosphonate oligonucleotide and then incubated for 5 hours at room temperature. The solution was removed and the support was washed twice with methanol, then reverse-flushed with argon.
V. General Procedure for Phosphorylation using Tributylammonium Pyrophosphate 1. 0.25 to 4 μmol synthesis scale To 0.25 to 4 μmol of the appropriate solid-supported 5’-phosphoroimidazolidate ON in a 1-
μmol Twist column (Glen Research) was added 1 mL of 0.5 M tris-tributylammonium pyrophosphate (Aldrich/or in-house prepared). The solution was pushed back and forth through the synthesis column for 5 min, then left to react for 17 h at room temperature. The solution was removed and the support was washed several times with methanol and acetonitrile, followed by a flush with argon. 2. Phosphorylation at the 10 μmol synthesis scale To 10 μmol of the appropriate solid-supported 5’-phosphoroimidazolidate ON in a 10-μmol Twist column (Glen) was added 10 mL of 0.5 M tris-tributylammonium pyrophosphate (Aldrich/or in-house prepared). The solution was pushed back and forth through the synthesis column for 5 min, then left to react for 17 h at room temperature. The solution was removed and the support was washed several times with methanol and acetonitrile, followed by a flush with argon.
3. Phosphorylation of 5’-Phosphoroimidazolidate ONs by Microwave Activation The phosphorylation reaction was performed using an Initiator microwave synthesizer from Biotage. The solid-supported 5’-phosphoroimidazolidate ONs (0.25 to 0.5 μmol) was placed in a 0.2 – 0.5 mL reaction vial (Biotage); 0.2 mL of 0.5 M tris-tributylammonium pyrophosphate was added, a stirring bar was added, and the mixture was magnetically stirred for 10 seconds and the vial was hermetically closed under argon. The vial was placed in the microwave synthesizer and the reaction was stirred for 40 minutes with microwave activation
S7
at 60 °C. At the end of the program, the reaction mixture was introduced into an oligonucleotide synthesis column, and the solid-supported ON TP was rinsed with methanol and acetonitrile and then flushed with argon several times. The supported ON TPs were then involved in deprotection steps.
4. Preparation of tris(tri-n-butylammonium) hydrogen pyrophosphate The corresponding tris(tetra-n-butylammonium) salt, suitable for phosphorylation reactions, is commercially available (Aldrich, Catalogue No. 93397). Otherwise, it can be easily prepared according to a previously described procedure 2 : 2.23 g (5 mmol) of tetra sodium pyrophosphate (ACS reagent, Sigma) were dissolved in 50 mL of milliQ water and eluted through a glass column filled with 20 mL of wet DOWEX-50WX8 resin, H+ from (Aldrich). After ion-exchange passage through the column, the solution was collected in a 250 mL flask, containing 20 mL of absolute ethanol (Carlo Erba) and 2.5 mL of tri-n-butylamine (Alfa Aesar), stirred at 0 °C. The column was rinsed with 50 more mL of water (outlet pH 5 to 6). The solvents were evaporated from the collected solution and the residue was then coevaporated four times with absolute ethanol. The residue was lyophilized from water, then from water/dioxane, and dried under vacuum over P2O5, affording tris(tri-n-butylammonium) hydrogen pyrophosphate as a white hygroscopic powder (2.8 g, 76%). The compound should present a single peak on its 31P NMR spectrum (121 MHz, CD3CN) at approximately -11 ppm (Figure S1); it can be stored for several months as a solid at -20 °C. The corresponding 0.5 M TBAPP solution in anhydrous DMF (Aldrich) was prepared after diluting 0.35 g of TBAPP per 1 mL of DMF, and stored over activated 3 Å molecular sieves for 2 days at 4 °C. No significant difference was found using either the commercial or the in-house reagents.
VI. Processing of DNA TPs
DNA TPs were deprotected as follows: 1.5 mL of 28% ammonia solution (Prolabo) was pushed back and forth through the synthesis column using two plastic syringes for 5 min and left to react for 1 hour. The solution was collected into a clean flask. An additional 1 mL of fresh ammonia was then pushed through the column and allowed to react for 30 minutes; this solution was collected in the same flask. This was repeated. In total, the DNA TP was treated with 3.5 mL of ammonia, in 3 steps over 2 hours. The ammonia was removed from the flask under reduced pressure and the residue was co-evaporated three times with water until
S8
dryness. The residue was dissolved in 1 mL of water, transferred to a 2-mL Eppendorf vial and concentrated in vacuo to a volume of 0.75 mL. Polythymidine TPs were directly lyophilized from water. Heteropolymeric DNA TPs were extracted 5 times with ether as follows: 0.75 mL of ether was added to the Eppendorf vial and the vial was closed and vortexed on a Vortex Genie-2 apparatus (Scientific Industries) for 1 minute. The vial was then centrifuged in a tabletop centrifuge (Sigma 1-13) at 6 000 min-1 for 2 minutes. The ether phase was removed. After extraction, the DNA TPs were lyophilized from water. Lyophilized DNA TPs were stored at -20 °C for several months without apparent degradation. The products at various steps in the synthesis of DNA TPs were monitored by IEX-HPLC as shown in Figure S1.
A
ppp
hp pp
a
p OH(dT)7 0
b
10
B
20 Minutes
30
ppp
hp p
pp
a
OH[d(TCTATGT)] 0
10
40
b 20 Minutes
30
40
Figure S1. Monitoring of the products at various steps in synthesis of TPsteps. (A) IEXHPLC (Method I.1.A) profiles for the phosphitylation reaction (Section III) of crude hp(dT)7 in green and profile of crude hp[ d(TCTATGT)] the products of oxidation/phosphorylation reactions (Sections IV and V) in blue. (B) IEX-HPLC profile of crude ppp[d(TCTATGT)] in green and the products of oxidation/phosphorylation reactions (Sections IV and V) in blue. These data demonstrate a conversion of about >95% at the phosphitylation step and >80% for the subsequent oxidation/phosphorylation step. Abbreviations: hp stands for 5’-Hphosphonate; p for 5’-monophosphate; pp for 5’-diphosphate; ppp for 5’-triphosphate. The different species were identified by IEX-LC/MALDI MS (see I.3).
S9
VII. Processing of 2’-O-TBDMS and 2’-O-Me RNA TPs
1. Deprotection of 2’-O-TBDMS and 2’-O-TBDMS/2’-O-Me protected RNA TPs RNA TPs were placed in an empty 15 mL plastic vial (or in a 50-mL vial for the 10-μmol scale). Three mL (or 10 mL for the 10-μmol scale) of cold 30% NH4OH (JTBaker or Prolabo) and absolute ethanol (– 3:1, v/v) were added and mechanically shaken for 2 hours at room temperature. The solution was filtered through a 0.45-μm filter (Nylon, VWR), the vial was rinsed, and the filtrates were freeze-dried. Deprotection of the 2’-O-TBDMS groups was performed as follows: The lyophilized material was dissolved in 0.5 mL 1 M TBAF (Aldrich, in THF solution, freshly opened bottle). An alternative desilylation method using 3HF-NEt3 and N-methylpyrrolidone at 65 oC resulted in significant to total loss of the triphosphate, (Figure S2. The solution was vortexed until complete dissolution and then left to react for 24 h at room temperature. It was then diluted with sterile water and purified by chromatography. For U7 TP, the solution was diluted to 2 mL with sterile water and eluted through a homemade ion-exchange DOWEX-50WX8-NH4+ column. The solution was collected, evaporated to dryness and desalted on a RP-C18 Poly-Pak II cartridge (Glen Research), first washed with 10 mM triethylammonium acetate (TEAAc) and then the ON was eluted with 50% acetronitrile in 10 mM TEAAc. The eluent was evaporated to dryness, transferred to a 2 mL Eppendorf vial, and lyophilized from water. For 21-mer RNA TPs, the reaction mediasolution was diluted to 15 mL (or to 30 mL for the 10 μmol scale) with sterile water and then purified using ion exchange semi-preparative chromatography, followed by semi-preparative reverse phase desalting, both performed on ÄKTApurifier purifying system (GE Healthcare), monitored at 260 nm, and using the Unicorn Software. Conditions were: IEX-HPLC semi prep: column: SP2019 DNA Pac PA100, 22 × 250 mm (Dionex); Buffer A: [25 mM TRIZMA-HCl (pH = 8.0, Aldrich), 5 M urea (Aldrich), 10% acetonitrile (Honeywell B&J, HPLC grade)]; Buffer B: [25 mM TRIZMAHCl 5 M urea (Aldrich), 10% acetonitrile (Honeywell B&J, HPLC grade), 0.5 M NaClO4 (Aldrich)]; Gradient: from 100% A to 50% B in 5 CV; flow 10 mL/min. The fractions containing ON were pooled and re-loaded onto the ÄKTApurifier for desalting. The conditions were as follows: RP-HPLC semi prep: column: XTerra C18, 30 × 150 mm (Waters); Buffer A: [25 mM TEAB (Aldrich), 10% acetonitrile (Honeywell B&J, HPLC grade)]; Buffer B: acetonitrile (Honeywell B&J, HPLC grade); Gradient: 100% A to 50% of B in 5 CV; flow 15 mL/min). The ON fractions were pooled and freeze-dried.
S10
Lyophilized RNA TPs were stored at -20 °C for several months without any degradation.
2. Deprotection of 2’-O-Me RNA TPs Appropriate RNA TPs were placed in a 15-mL screw cap plastic vial. Three mL of cold 30% NH4OH (JTBaker) and absolute ethanol (–3:1, v/v) were added and the solution was incubated for 2 hours at room temperature with shaking. The solution was filtered through a 0.45-μm filter (Nylon, VWR), the vial was rinsed and the filtrates were freeze-dried. The crude ONs were dissolved in water and loaded on the ÄKTApurifier and purified using a RPHPLC semi-prep: column using the following conditions: XTerra C18, 30 × 150 mm (Waters); Buffer A: [25 mM TEAB (Aldrich), 10% acetonitrile (Honeywell B&J, HPLC grade)]; Buffer B: acetonitrile (Honeywell B&J, HPLC grade); Gradient: 100% A to 50% of B in 5 CV; flow 15 mL/min). The fractions containing ON were pooled and freeze-dried. ppp
2320.05 2236.73 2158.52
A
pp
1087.73
p
0
10
20 Minutes
2433.87
30
40 1000
Mass (m/z)
4000
2320.65
ppp
B 2237.10 2160.36
pp 661.56
p 0
10
20 Minutes
30
40 1000
2556.79
Mass (m/z)
4000
Figure S2. Hydrolysis of the 5’-triphosphate moiety by fluoride ion treatment. (A) IEXHPLC and MALDI/TOF-MS profiles for pppU7 treated with 3HF-NEt3 at 65 oC for 1.5 h. (B) IEX-HPLC and MALDI/TOF-MS profiles for pppU7 treated with 1 M TBAF at room temperature for 24 h. Calculated [M-H]- m/z values: pU7: 2160.60; ppU7: 2240.35; pppU7: 2320.65.
S11
VIII. Processing of 2’-O-PivOM RNA TPs
1. Deprotection of U7 TP CPG supported U7 TP in the oligonucleotide synthesis column, was deprotected as follows: It was first treated with 1 M anhydrous 1,8-diazabicyclo-[5,4,0]undec-7-ene (DBU, Fluka) in acetonitrile at room temperature for 1 min to eliminate the cyanoethyl groups from phosphates. Then the DBU solution was removed from the column and the solid support was washed four times with 3 mL anhydrous acetonitrile. Secondly, a 28% aqueous ammonia solution was applied to the column in three batches (1.5 mL, 1 mL, 0.5 mL) for 0.5 h each (see Section VI). The three ammonia filtrates were pooled in a screw-capped plastic vial and were left at room temperature for 1.5 hour more to completely deprotect 2’-hydroxyl protecting groups. The fully deprotected oligonucleotide was transferred to a 50-mL roundbottomed flask and evaporated to dryness. The residue was co-evaporated three times with water. The residue was dissolved in 1 mL of water, transferred to a 2-mL Eppendorf vial and lyophilized from water.
2. Deprotection of RNA TPs CPG-supported RNA TPs of mixed base were placed in an oligonucleotide synthesis column and deprotected as described in Section VII.1. The fully deprotected oligoribonucleotide TP was transferred to a 50-mL round-bottomed flask. Isopropylamine (15% of total volume: 0.45 mL) was added to the solution before evaporation to dryness. The residue was co-evaporated three times with water, dissolved in 1 mL of water, transferred to a 2-mL Eppendorf vial and extracted five times with ether. After extraction, the RNA TP was lyophilized from water.
S12
IX. Table S1: Summary for the Prepared Oligonucleotide TPs HPLC OD260 Scale MALDI-Tof MS Synthesis purity Yield (negative mode) Conditionsc (µmol) d e (%) (%) 22.2 Calcd: 2306.35, 30 h, rt 0.5 82.5 73% Found: 2306.66 18.0 Calcd: 2306.35, 17 h, rt 0.5 83.8 60% Found: 2306.10 9.0 Calcd: 2306.35, 2 h MW, 60 0.25 81.9 °C 60% Found: 2306.67 7.5 Calcd: 2306.35, 1 h MW, 60 0.25 81.5 °C 50% Found: 2306.99 40 min MW, 20.0 Calcd: 2306.35, 0.5 83.4 66% Found: 2306.82 60 °C 24.0 Calcd: 2306.35, 20 min MW, 0.5 80.5 60 °C 79% Found: 2306.93 28.0 Calcd: 2325.36, 17 h, rt 0.5 83.8 81% Found: 2325.70 40 min MW, 25.0 Calcd: 2325.36, 0.5 82.6 72% Found: 2325.48 60 °C 40 min MW, Calcd: 2320.17, 60 °C; 0.25 57.8 n.d. Found: 2320.05 Et3N-3HF 40 min MW, 5.2 Calcd: 2320.17, 0.25 74.6 60 °C; 33% Found: 2320.51 TBAF
Entry
ONTP sequence
1
ppp(dT)7
2
ppp(dT)7
3
ppp(dT)7
4
ppp(dT)7
5
ppp(dT)7
6
ppp(dT)7
7
ppp[d(TCTATGT)]
8
ppp[d(TCTATGT)]
9
pppU7a
10
pppU7a
11
pppUUGUCUCUGGUCCUUACUUAAa,f
17 h, rt, TBAF
2.0
93.2
108.5 26%
Calcd: 6787.87, Found: 6790.06
12
pppUUGUCUCUGGUCCUUACUUAAa,f
17 h, rt, TBAF
10.0
81.2
560.0 30%
Calcd: 6787.87, Found: 6793.44
13
pppAccGAAGuGuuGuuuGuccdTsdTa,f
17 h, rt TBAF
1.0
89.2
61.4 32%
Calcd: 7033.35Found: 7035.48
14
pppaaguaaggaccagagacaadTsdT
17 h, rtb
4.0
77.0
375.2 40%
Calcd: 7307.84Found: 7310.93
15
pppU7b
40 min MW, 60 °C
0.25
77.3
10.5 66%
Calcd: 2320.17, Found: 2320.47
16
pppAGUUGUUCCCb
17 h, rt
0.5
77.2
24.0 50%
Calcd: 3336.84, Found: 3335.59
a
Prepared using 2’-O-TBDMS protected phosphoramidites. Prepared using 2’-O-PivOM protected phosphoramidites. c Conditions for phosphorylation reaction (see Section V). d Percentage of triphosphate as calculated from integration of the major peak in the crude mixture ion-exchange chromatogram. e Isolated yield of the crude material. f Purified full-length material. Abbreviations: d = 2'-deoxy; upper case = 2'-OH; lower case = 2'-O-methyl; s = phosphorothioate; ppp = 5'triphosphate. b
S13
XI. Supplementary Experimental Figures
Figures depict experimental data for ON TPs from Table S1 and for some intermediate 5’-Hphosphonates.
IEX-HPLC (Methods described in Section I.1) at 260 nm of crude products (unless specified for HPLC purified products) with tR (min) and % of triphosphate given. Negative mode MALDI-Tof MS of crude products (unless specified for HPLC purified products) with Calculated and Found m/z.
NMR data (Methods described in Section I.4). O -O P hp = H
ppp =
O O O -O P O P O P OOO-
S14
0,46 0,44
HPLC (Method A) – tR = 19.5 min / 94.8%
0,42 0,40 0,38
hp(dT)7
0,36 0,34 0,32 0,30 0,28 0,26
0,22 0,20 0,18 0,16 0,14 0,12 0,10 0,08 0,06 0,04 0,02 0,00 -0,02 0,00
2,00
4,00
6,00
8,00
10,00
12,00
14,00
16,00
18,00
20,00 Minutes
22,00
24,00
26,00
28,00
30,00
32,00
34,00
36,00
2130.41
100
90
38,00
70
60
50
40
30
20 1064.99
2066.01
10
0 800
1440
2080
2720 Mass (m/z)
S15
40,00
1.2E+4
MALDI – m/z Calcd : 2130.40, Found : 2130.41
80
% Intensity
AU
0,24
3360
0 4000
0,170 0,160 0,150 0,140 0,130
ppp(dT)7 Table S1, entry 1 0.5 µmol/30 h rt
HPLC (Method A) – tR = 25.6 min / 82.5%
0,120 0,110 0,100
AU
0,090 0,080 0,070 0,060 0,050 0,040 0,030 0,020 0,010 0,000
0,00
2,00
4,00
6,00
8,00
10,00
12,00
14,00
16,00
18,00
20,00 Minutes
22,00
24,00
26,00
28,00
30,00
32,00
34,00
2306.66
100
36,00
38,00
2.6E+4
90
MALDI – m/z Calcd : 2306.35, Found : 2306.66
80
% Intensity
70
60
50
40
30 2222.71 20
1153.06 2066.652236.58 2344.46
10
0 800
1440
2080
2720 Mass (m/z)
S16
40,00
3360
0 4000
0,36 0,34 0,32 0,30 0,28
ppp(dT)7 Table S1, entry 2 0.5 µmol/17 h rt
HPLC (Method A) – tR = 25.7 min / 83.8%
0,26 0,24 0,22 0,20
AU
0,18 0,16 0,14 0,12 0,10 0,08 0,06 0,04 0,02 0,00 -0,02 0,00
2,00
4,00
6,00
8,00
10,00
12,00
14,00
16,00
18,00
20,00 Minutes
22,00
24,00
26,00
28,00
30,00
32,00
34,00
2306.10
100
36,00
38,00
2.4E+4
90
MALDI – m/z Calcd : 2306.35, Found : 2306.10
80
% Intensity
70
60
50
40
30 2066.31 20
1152.85
2366.37
1163.52 0 800
2298.29
2225.77 2344.46 2144.81
10
1440
2080
2720 Mass (m/z)
S17
40,00
3360
0 4000
0,34 0,32 0,30 0,28 0,26
ppp(dT)7 Table S1, entry 3 0.25 µmol/2 h MW, 60 oC
HPLC (Method A) – tR = 25.6 min / 81.9%
0,24 0,22 0,20
AU
0,18 0,16 0,14 0,12 0,10 0,08 0,06 0,04 0,02 0,00
0,00
2,00
4,00
6,00
8,00
10,00
12,00
14,00
16,00
18,00
20,00 Minutes
22,00
24,00
26,00
28,00
30,00
32,00
34,00
36,00
2306.67
100
38,00
40,00
6807
MALDI – m/z Calcd : 2306.35, Found : 2306.67
90
80
% Intensity
70
60
50
40
2298.33
30 2066.04 20
1152.89 2145.662328.29 2344.25 2222.11 2368.27
10
0 800
1440
2080
2720 Mass (m/z)
S18
3360
0 4000
0,32 0,30 0,28 0,26 0,24
ppp(dT)7 Table S1, entry 4 0.25 µmol/1 h MW, 60 oC
0,22
HPLC (Method A) – tR = 25.7 min / 81.5%
0,20
AU
0,18 0,16 0,14 0,12 0,10 0,08 0,06 0,04 0,02 0,00
0,00
2,00
4,00
6,00
8,00
10,00
12,00
14,00
16,00
18,00
20,00 Minutes
22,00
24,00
26,00
28,00
30,00
32,00
34,00
2306.99
100
36,00
38,00
40,00
1.9E+4
MALDI – m/z Calcd : 2306.35, Found : 2306.99
90
80
% Intensity
70
60
50
40
30 2226.44 2066.84
20
10
0 799.0
2329.00
1153.12
1439.4
2079.8
2720.2 Mass (m/z)
S19
3360.6
0 4001.0
0,34 0,32 0,30 0,28 0,26
ppp(dT)7 Table S1, entry 5 0.5 µmol/40 min MW, 60 oC
HPLC (Method A) – tR = 24.9 min / 83.4%
0,24 0,22 0,20
AU
0,18 0,16 0,14 0,12 0,10 0,08 0,06 0,04 0,02 0,00
0,00
2,00
4,00
6,00
8,00
10,00
12,00
14,00
16,00
18,00
20,00 Minutes
22,00
24,00
26,00
28,00
30,00
32,00
34,00
2306.82
100
36,00
38,00
40,00
3.8E+4
90
MALDI – m/z Calcd : 2306.35, Found : 2306.82
80
% Intensity
70
60
50
2299.37
40
30
20 1153.03
2146.19 2344.57 2237.16
10
0 799.0
1439.4
2079.8
2720.2 Mass (m/z)
S20
3360.6
0 4001.0
0,32 0,30 0,28 0,26 0,24
ppp(dT)7 Table S1, entry 6 0.5 µmol/20 min MW, 60 oC
HPLC (Method A) – tR = 24.9 min / 80.5%
0,22 0,20
AU
0,18 0,16 0,14 0,12 0,10 0,08 0,06 0,04 0,02 0,00
0,00
2,00
4,00
6,00
8,00
10,00
12,00
14,00
16,00
18,00
20,00 Minutes
22,00
24,00
26,00
28,00
30,00
32,00
34,00
2306.93
100
36,00
38,00
40,00
1.9E+4
MALDI – m/z Calcd : 2306.35, Found : 2306.93
90
80
% Intensity
70
60
50
40
2299.33
30
20
1153.18 2066.96 2328.98
10
2222.86 1149.35
0 799.0
2002.52 1439.4
2079.8
2720.2 Mass (m/z)
S21
3360.6
0 4001.0
0,32 0,30
Crude ppp(dT)7
0,28 0,26
HPLC (Method A) – tR = 25,8 min / 81%
0,24 0,22 0,20
AU
0,18 0,16
0,14 0,12
0,10 0,08
0,06 0,04
0,02 0,00
0,00
2,00
4,00
6,00
8,00
10,00
12,00
14,00
16,00
18,00
20,00 Minutes
22,00
24,00
26,00
28,00
30,00
32,00
34,00
36,00
38,00
40,00
0,34
HPLC purified ppp(dT)7
0,32
0,30
0,28
0,26
HPLC (Method A) – tR = 24.7 min / 90%
0,24
0,22
0,20
0,16
0,14
0,12
0,10
0,08
0,06
0,04
0,02
0,00
0,00
2,00
4,00
6,00
8,00
10,00
12,00
14,00
16,00
18,00
20,00 Minutes
22,00
24,00
26,00
28,00
30,00
32,00
34,00
2306.71
100
36,00
38,00
40,00
4929.0
90
MALDI – m/z Calcd : 2306.35, Found : 2306.71
80 % Intensity
AU
0,18
70 2328.83
60 50 40
2344.69
30
2366.68
20
2387.89 2226.49
10 0 799.0
1439.2
2079.4
2719.6 Mass (m/z)
S22
3359.8
0 4000.0
0,190 0,180
hp[d(TCTATGT)]
0,170 0,160
HPLC (Method A) – tR = 20.6 min / 94.5%
0,150 0,140 0,130 0,120 0,110
AU
0,100 0,090 0,080 0,070 0,060 0,050 0,040 0,030 0,020 0,010 0,000
0,00
2,00
4,00
6,00
8,00
10,00
12,00
14,00
16,00
18,00
20,00 Minutes
22,00
24,00
26,00
28,00
30,00
32,00
34,00
2149.69
100
36,00
38,00
1.5E+4
90
MALDI – m/z Calcd : 2149.40, Found : 2149.69
80
% Intensity
70 60 50 40 30 20 2171.50 10 0 800
4301.21
2158.88 2208.73
1074.66 1640
40,00
2480
3320 Mass (m/z)
S23
4160
0 5000
0,55
ppp[d(TCTATGT)]
0,50
0,45
HPLC (Method A) – tR = 25.9 min / 83.8%
Table S1, entry 7 0.5 µmol/17 h rt
0,40
0,35
AU
0,30
0,25
0,20
0,15
0,10
0,05
0,00
0,00
2,00
4,00
6,00
8,00
10,00
12,00
14,00
16,00
18,00
20,00 Minutes
22,00
24,00
26,00
28,00
30,00
32,00
34,00
2325.70
100
36,00
38,00
40,00
2.9E+4
90
MALDI – m/z Calcd : 2325.36 ; Found : 2325.70
80
% Intensity
70
60
50
40
2317.73
30 2165.27 20 2085.48 2241.15 2363.49
1162.71 10
1571.75 1188.09
0 799.0
1439.4
2307.05 2148.57
2079.8
2720.2 Mass (m/z)
S24
3360.6
0 4001.0
0,34 0,32
ppp[d(TCTATGT)] HPLC (Method A) – tR = 25.5 min / 82.6%
0,30 0,28 0,26
Table S1, entry 8 0.5 µmol/40 min MW, 60 oC
0,24 0,22 0,20
AU
0,18 0,16 0,14 0,12 0,10 0,08 0,06 0,04 0,02 0,00
0,00
2,00
4,00
6,00
8,00
10,00
12,00
14,00
16,00
18,00
20,00 Minutes
22,00
24,00
26,00
28,00
30,00
32,00
34,00
2325.48
100
36,00
38,00
40,00
4.1E+4
90
MALDI – m/z Calcd : 2325.36 ; Found : 2325.48
80
% Intensity
70
60
50 2317.63 40
30
20 1162.29
1571.86
10
0 799.0
1439.4
1851.22
2165.04 2084.87 2241.20 2363.45
2079.8
2720.2 Mass (m/z)
S25
3360.6
0 4001.0
2829.67
100
90
9.6E+3
hp[(UTBDMS) 6U] MALDI – m/z Calcd : 2829.77, Found : 2829.67
80
70
% Intensity
60
50
40 2851.76 30
20
2867.71
1083.30
10
2889.17 2765.49
1414.03 2345.10
0 799.0
1439.2
2079.4
2985.61
2719.6
3359.8
Mass (m/z) 2144.80
100
90
2715.10
0 4000.0 6171.9
hpU7
80
MALDI – m/z Calcd : 2144.21, Found : 2144.80
% Intensity
70
60
50
40 1468.03 1774.01
30 1161.96 20 855.70 847.62
2259.17
10
0 799.0
1439.2
2079.4
2719.6 Mass (m/z)
S26
3359.8
0 4000.0
0,20
pppU7
0,18
HPLC (Method A) – tR = (ppp): 25.4 min / 57.8% (pp): 23.6 min / 23.7% (p): 21.6 min / 12.6%
Table S1, entry 9
0,16
0.25 μmol/40 min MW at 60 °C
0,14
0,10
90 min Et3N-3HF/Et3N/NMP
0,08
65 °C
0,06
0,04
0,02
0,00
-0,02
-0,04
0,00
2,00
4,00
6,00
8,00
10,00
12,00
14,00
16,00
18,00
20,00 Minutes
22,00
24,00
26,00
28,00
30,00
32,00
34,00
2320.05
100
36,00
38,00
40,00
1962.1
2236.73 90
2148.52
80
70
1087.73
60 % Intensity
AU
0,12
2243.50 50
2324.48
40
MALDI – m/z Calcd : 2320.17, Found : 2320.05
2433.87
30
2157.97 2314.40
20
10
0 999.0
1599.2
2199.4
2799.6 Mass (m/z)
S27
3399.8
0 4000.0
0,26
0,24
0,22
0,20
0,18
pppU7 Table S1, entry 10
HPLC (Method A) – tR = 24.8 min / 74.6%
0.25 μmol/40 min MW at 60 °C 24 h TBAF rt
AU
0,16
0,14
0,12
0,10
0,08
0,06
0,04
0,02
0,00
0,00
2,00
4,00
6,00
8,00
10,00
12,00
14,00
16,00
18,00
20,00 Minutes
22,00
24,00
26,00
28,00
30,00
32,00
2320.51
100
34,00
36,00
38,00
1.7E+4
90
MALDI – m/z Calcd : 2320.17, Found : 2320.51
80
70
% Intensity
2240.33 60
50
40
2342.57
30
2161.35 2262.34
20 855.61
1161.63 1467.71
10
1774.55 0 599.0
1279.2
2080.28
1959.4
2639.6 Mass (m/z)
S28
40,00
3319.8
0 4000.0
0,30
0,28
0,26
0,24
0,22
pppU7 HPLC (Method A) – tR = 25.4 min / 77.3%
Table S1, entry 15 0.25 μmol/40 min MW at 60 °C
0,20
0,18
AU
0,16
0,14
0,12
0,10 0,08
0,06
0,04
0,02
0,00
0,00
2,00
4,00
6,00
8,00
10,00
12,00
14,00
16,00
18,00
20,00 Minutes
22,00
24,00
26,00
28,00
30,00
32,00
2320.47
100
34,00
36,00
38,00
2.4E+4
90
MALDI – m/z Calcd : 2320.17, Found : 2320.47
80
70
% Intensity
60
50
40
30 1160.00 20
10
0 800
2240.98 2209.19 2080.82 1468.13
1440
1774.16
2080
2720 Mass (m/z)
S29
40,00
3360
0 4000
0,40 0,38 0,36 0,34 0,32 0,30 0,28
pppAGUUGUUCCC Table S1, entry 16
HPLC (Method A) – tR = 21.4 min / 77.2%
0.5 μmol/rt 17 h
0,26 0,24
AU
0,22 0,20 0,18 0,16 0,14 0,12 0,10 0,08 0,06 0,04 0,02 0,00 -0,02 0,00
2,00
4,00
6,00
8,00
10,00
12,00
14,00
16,00
18,00
20,00
22,00
24,00 26,00 Minutes
28,00
30,00
32,00
34,00
36,00
38,00
40,00
42,00
3335.59
100
44,00
46,00
48,00
1.7E+4
90
MALDI – m/z Calcd : 3336.84, Found : 3335.59
80
70
% Intensity
60
50 3374.21 40
30
3175.79 3029.85 3253.06
20
10
0 1000
1800
2600
3400 Mass (m/z)
S30
50,00
4200
0 5000
hpUUGUCUCUGGUCCUUACUUAA Synthesis crude 0.30
HPLC (Method B) – tR = 19.7 min / 56.2%
AU
0.20
0.10
0.00 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 Minutes
0.40
pppUUGUCUCUGGUCCUUACUUAA Synthesis crude
HPLC (Method B) – tR = 19.6 min / 59.2%
AU
0.30
0.20
0.10
0.00 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 Minutes
S31
0.30
HPLC (Method B) – tR = 19.8 min / 95.2%
AU
pppUUGUCUCUGGUCCUUACUUAA HPLC purified full-length Table S1, entry 11 0.20 2 μmol/rt 17 h 0.10
0.00 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 Minutes 0.20
HPLC (Method C) – tR = 24.8 min / 93.2%
AU
0.15
0.10
0.05
0.00 0.00
5.00
10.00
15.00
20.00 Minutes
30.00
35.00
6790.06
100 90
40.00
1.9E+4
MALDI – m/z Calcd : 6787.87, Found : 6790.06
80 70 % Intensity
25.00
60 50 6812.00
40 30 20
6709.49 6614.27
3396.09
10 0 2999.0
4399.4
5799.8
Mass (m/z)
S32
7200.2
8600.6
0 10001.0
pppUUGUCUCUGGUCCUUACUUAA 0.50
AU
0.40
HPLC purified full-length Table S1, entry 12 10 μmol/rt 17 h
HPLC (Method B) – tR = 18.5 min / 81.2%
0.30
0.20
0.10
0.00 0.00
5.00
10.00
15.00
0.18
20.00 Minutes
25.00
30.00
35.00
40.00
0.16
HPLC (Method C) – tR = 24.9 min / 81.8%
0.14 0.12
AU
0.10 0.08 0.06 0.04 0.02 0.00 0.00
5.00
10.00
15.00
20.00 Minutes
25.00
30.00
35.00
6793.44
100
40.00
1.1E+4
MALDI – m/z Calcd : 6787.87, Found : 6793.44
90 80
% Intensity
70 60 50 40 30 20
6713.18
3397.94
6617.18
10 0 2999.0
4399.4
5799.8
Mass (m/z)
S33
7200.2
8600.6
0 10001.0
hpAccGAAGuGuuGuuuGuccdTsdT 0.30 Synthesis crude
HPLC (Method B) – tR = 19.7 min / 54.3%
AU
0.20
0.10
0.00 0.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
40
Minutes
pppAccGAAGuGuuGuuuGuccdTsdT Synthesis crude
HPLC (Method B) – tR = 19.6 min / 52.2%
0.40
AU
0.30
0.20
0.10
0.00 0.00
5.00
10.00
15.00
20.00 Minutes
S34
25.00
30.00
35.00
40
0.25
0.20
pppAccGAAGuGuuGuuuGuccdTsdT HPLC purified full-length Table S1, entry 13 1 μmol/rt 17 h
HPLC (Method B) – tR = 18.8 min / 92.8%
AU
0.15
0.10
0.05
0.00 0.00
5.00
10.00
15.00
20.00 Minutes
25.00
30.00
35.00
40.00
0.25
HPLC (Method C) – tR = 23.9 min / 89.2%
0.20
AU
0.15
0.10
0.05
0.00 0.00
5.00
10.00
15.00
20.00 Minutes
25.00
30.00
35.00
7035.48
100
40.00
1.6E+4
MALDI – m/z Calcd : 7033.35, Found : 7035.48
90 80
% Intensity
70 60 50 40 30
3519.15
6955.56 6795.47
20 6146.92 10 0 2999.0
4399.4
5799.8
6874.5 4 6635.93
Mass (m/z)
S35
7200.2
8600.6
0 10001.0
pppaaguaaggaccagagacaadTsdT 0.06 Table S1, entry 14 4 μmol/rt 17 h
HPLC (Method B) – tR = 19.0 min / 79.8%
AU
0.04
0.02
0.00 0.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
Minutes
HPLC (Method C) – tR = 24.9 min / 77.0%
0.006
AU
0.004
0.002
0.000 0.00
5.00
10.00
15.00
20.00 Minutes
100
25.00
30.00
35.00
7310.93
40.00 7909.0
MALDI – m/z Calcd : 7307.84, Found : 7310.93
90 80
% Intensity
70 7352.98
60 50
7332.25 40 3319.433638.65 3957.96 4300.73 3109.5 6 20 3084.38 4660.41
7231.52
30
7150.53
5705.11 6024.56
10 0 2999.0
4399.4
5799.8
Mass (m/z)
S36
7200.2
8600.6
0 10001.0
ppp(dT)7 (NMR Method A):
-4
-6
-8
-20
-40
-60
-80
-100
ppm
-22.629
0
-1 0
-1 2 0.94
-1.057 -1.094 -1.135 -2 6.02
20
-14
-16
-18
-20
- 22
ppm 0.97
40
-11.394 -11.549
60
1.00
80
-10.384 -10.525
100
S37
pppaaguaaggaccagagacaadTsdT Table S1, entry 14 (NMR Method B):
S38
pppUUGUCUCUGGUCCUUACUUAA Table S1, entry 12 (NMR Method B):
S39
