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Supporting Information
Metallophyte wastes and polymetallic catalysis: a promising combination in green chemistry. The illustrative synthesis of 5’-capped RNA
Yann Thillier,[a] Guillaume Losfeld,[b] Christelle Dupouy,[a] Vincent Escande,[b] Jean-Jacques Vasseur,[a] Françoise Debart,*[a] and Claude Grison*[b]
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MATERIALS AND METHODS
Carbon tetrachloride was distilled under phosphorus pentoxide. Pyridine, acetonitrile,
triethylamine, butylamine and tri-n-butylamine were distilled under calcium hydride. Solvents
and other reagents were purchased from Aldrich and used without further
purifications. Analytical and semi-preparative high performance liquid chromatographies
were performed on a Dionex DX 600 HPLC system or a Dionex U 3000 HPLC system
equipped with anion-exchange DNAPac PA 100 columns (4 x 250 mm for analysis or 9 x 250
mm for purification, Dionex). The following HPLC solvent systems were used: 5% CH3CN in
25 mM Tris-HCl buffer, pH 8 (buffer A) and 5% CH3CN containing 400 mM NaClO4 in 25 mM
Tris-HCl buffer, pH 8 (buffer B). Flow rates were 1.5 mL.min-1 and 5 mL.min-1 for analysis and
semi-preparative purposes, respectively. MALDI-TOF mass spectra were recorded on a
Voyager-DE spectrometer (Perseptive Biosystems, USA) 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. Analytical samples were mixed with the matrix in a 1:5 (v/v) ratio,
crystallized on a 100-well stainless steel plate and analyzed. UV quantitation of RNAs was
performed on a Varian Cary 300 Bio UV/Visible spectrometer by measuring absorbance at
260 nm.
The solid-supported 5’-H-phosphonate 2 and 5’-phosphoroimidazolide 3 derivatives, as well
as GpppRNAs were synthetized as previously described (Y. Thillier and al. RNA 2012, 18,
856-868).
Synthesis of T6 and RNA on solid-support
Oligonucleotides T6 and RNA synthesis was performed on an ABI 394 synthesizer (Applied
Biosystems) at 1 mol scale from commercially available (Link Technologies) long chain
alkylamine controlled-pore glass (LCAA-CPG) solid support with a pore size of 1000 Å
derivatized through the succinyl linker with 5’-O-dimethoxytrityl-[thymidine, 2’-O-acetyl-N6-
phenoxyacetyl adenosine or 2’-O-acetyl-N2-dimethylformamide guanosine]. T6 and RNAs
sequences were assembled on a 1 mol scale in Twist oligonucleotide synthesis columns
(Glen research) using 5’-O-DMTr-dT-3’-O-(O-cyanoethyl-N,N-diisopropyl-phosphoramidite)
from Pierce technologies; the PivOM amidites (5’-O-DMTr-2’-O-PivOM-[U, CAc, APac or
GiPrPac]-3’-O-(O-cyanoethyl-N,N-diisopropyl-phosphoramidite) and the 5’-O-DMTr-2’-O-Me-
APac-3’-O-(O-cyanoethyl-N,N-diisopropyl-phosphoramidite) were all purchased from
Chemgenes. The PrOM amidites (5’-O-DMTr-2’-O-PrOM-[U, APac ]-3’-O-(O-cyanoethyl-N,N-
diisopropyl-phosphoramidite) were prepared according to an unpublished procedure.
Phosphoramidites were vacuum dried prior to their dissolution in extra dry acetonitrile
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(Biosolve) at 0.1 M. For the coupling reaction, the activator was 5-benzylmercaptotetrazole
(BMT, Chemgenes) used at 0.3 M concentration. Dichloroacetic acid (3% in CH2Cl2) (Glen
research) was the detritylation reagent. The capping step was performed with a mixture of
5% phenoxyacetic anhydride (Pac2O) in THF and 10% N-methylimidazole in THF (Link
Technologies). The oxidizing solution was 0.1 M iodine in THF/pyridine/H2O (78:20:2; v/v/v)
(Link Technologies). After RNA assembly completion, the column was removed from the
synthesizer and dried under a stream of argon.
Capping reaction with GDP
Guanosine-5’-diphosphate sodium salt (0.5 g, 1.2 mmol) was purchased from Jena
Bioscience and before use it was converted into its tri-n-butylammonium salt by passing
through a glass column filled with 20 mL of wet DOWEX-50W X 8 resin, H+ form. It was
collected in a 250 mL round flask containing absolute ethanol (12 mL) and tri-n-butylamine
(0.72 mL), stirred at 0°C. The Dowex column was rinsed with water (80 mL) to reach pH 5 to
6. The solvents were evaporated from the collected solution and the residue was
coevaporated four times with absolute ethanol then was lyophilized from water to afford a
white hygroscopic powder (0.938 g, 1.15 mmol, 96%). The desired GDP tri-n-
butylammonium salt was characterized by two doublets at -10.55 and -11.20 ppm (31P-NMR,
121 MHz, D2O). It can be stored as a solid for several weeks at -20°C.
In a dry 2 mL microcentifuge tube (Costar), bis (tri-n-butylammonium) guanosine
diphosphate (103 mg, 0.14 mmol) and the freshly dehydrated catalytic extracts required for
the coupling (0.2 mmol) whose the amount was estimated from the concentration of the
metal (Zn or Ni) determined by ICP-MS (Table 1) were mixed in anhydrous DMF (0.5 mL) to
get a final concentration of 0.4 M in the capping solution. The tube was closed and the
mixture was vortexed for 5 minutes on a Top-Mix 1118 (Fischerbrand) and centrifuged in a
tabletop centrifuge (Sigma 1-13) at 6000 min-1 for 30 seconds. This was repeated twice.
Then the supernatant was taken using a glass syringe filled with 3 beads of 4Å molecular
sieves. Using another syringe, the solution was applied to the column containing the solid-
supported 5’-phosphoroimidazolidate oligonucleotide (1 mol), and left to react for 18 h at
30°C. The solution was removed and the support was washed with water (2 x 2 mL), a 0.1 M
aqueous solution of EDTA (pH 7, 2 x 2 mL), and dry CH3CN (4 x 2 mL). Finally the column
was dried by blowing argon through it during 1 min.
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Deprotection and release of GpppT6 (1-12) and GpppRNAs (13, 15, 16, 18, 19, 21)
The solid-supported GpppT6 and GpppRNAs were deprotected and released from the
support as follows: firstly, a 1 M solution of 1,8-diazadicyclo-[5,4,0]undec-7-ene (DBU) in
anhydrous CH3CN was applied to the column for 3 min. Then the solution was removed and
the solid-support was washed with anhydrous CH3CN. The support was dried by a 1 min
flush with argon. Secondly, a 30 % aqueous ammonia solution was applied to the column in
three batches (1.5 mL, 1mL, 0.5 mL) for 30 min each. The three ammonia fractions were
collected in a 4 mL screw-capped glass vial and were left to react at room temperature for
1.5 h. The fully deprotected oligonucleotides were transferred to 50 mL round-bottomed
flasks and isopropylamine (15% of total volume: 0.45 mL) was added only to the solutions of
GpppRNAs. Then the mixtures were evaporated under reduced pressure with a bath at 30°C
maximum until the volumes were reduced to 0.3 mL. The mixtures were coevaporated three
times with 1 mL of water following the same protocol. The residues were redissolved in water
(1.5 mL divided in three portions for flask rinse: 0.8 mL, 0.4 mL, 0.3 mL) and transferred to 2
mL Eppendorf-vials then lyophilized from water.
Analytical and purification of GpppT6 (1-12) and GpppRNAs (15, 18, 21)
The crude GpppT6 (1-12) and GpppRNAs 6-mers (15, 18, 21) were analyzed on a Dionex DX
600 HPLC system monitored at 260 nm with a 0%-30% linear gradient of buffer B in buffer A.
Only GpppRNAs (15, 18, 21) were purified using a 0%-30% linear gradient of buffer B in
buffer A. The fractions containing the pure 5’-capped oligonucleotides were pooled in a 100
mL round-bottomed flask and were concentrated to a volume of 0.5 mL under reduced
pressure with a bath at 30°C. Then the pure GpppRNAs were ready for the desalting
procedure.
Sephadex procedure for crude GpppT6 (5, 9)
The crude GpppT6 5 and 9 were dissolved in 1 mL of water, and were loaded on a NAP-10
Column (Sephadex G-25 DNA grade, GE healthcare). Oligonucleotides were eluted in a 2
mL Eppendorf-vial with 1.5 mL of water then lyophilized.
Capping reaction with 7mGDP
7-Methylguanosine-5’-diphosphate sodium salt (0.25 g, 0.5 mmol) was purchased from Jena
Bioscience and before use, it was converted into its tri-n-butylammonium following the same
procedure as above using 15 mL of wet DOWEX-50W X 8 resin, H+ form ; 10 mL of absolute
ethanol and tri-n-butylamine (0.3 mL, 1.26 mmol). The solvents were evaporated from the
collected solution and the residue was coevaporated four times with absolute ethanol then
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was lyophilized from water to afford a white hygroscopic powder (0.353 g, 0.47 mmol, 94 %).
The desired 7mGDP, tri-n-butylammonium salt was characterized by two doublets at -9.98
and -11.13 ppm (31P-NMR, 121 MHz, D2O). It can be stored as a solid for several days at -
20°C.
The capping reaction with bis (tri-n-butylammonium)-7mGDP was performed as previously
described. A two-fold decrease in the quantities of reagents used was applied in this case. In
a 2 mL Eppendorf vial, bis (tri-n-butylammonium)-7mGDP (50 mg, 0.07 mmol) and the correct
amount of freshly dehydrated catalytic extract were mixed in anhydrous DMF (0.25 mL). The
tube was closed and the mixture was vortexed for 5 minutes and centrifuged in a tabletop
centrifuge at 6000 min-1 for 30 seconds. This was repeated twice. Then the supernatant was
taken using a 1 mL plastic syringe filled with a bead of 4Å molecular sieves. The solution was
applied to the column containing the solid-supported 5’-phosphoroimidazolidate
oligonucleotide and pushed back and forth for 5 minutes then left to react for 18 h at 30°C.
The solution was removed and the support was washed with water (2 x 2 mL), a 0.1 M
aqueous solution of EDTA (pH 7, 2 x 2 mL), and dry CH3CN (4 x 2 mL). Finally the column
was dried by blowing argon through it during 1 min.
Deprotection and release of 7mGpppRNAs (14, 17, 20)
The solid-supported 7mGpppRNAs were deprotected and released from the support as
follows: it was first treated with a 1M solution of 1,8-diazadicyclo-[5,4,0]undec-7-ene (DBU) in
anhydrous CH3CN for 3 min. Then the solution was removed and the solid-support was
washed with anhydrous CH3CN. The support was reverse-flushed by a 1 min flush with
argon and dried under vacuum over P2O5 for 2 hours.
In a dry 10 mL round-bottomed flask, 2.7 mL of dry butylamine was added in 1.3 mL of dry
THF under argon. Then the Twist oligonucleotide synthesis column was opened and a half
quantity of the solid-support was transferred in another column. 2 mL of the butylamine / THF
(2:1) solution was applied to each column using two glass syringes filled of 4Å molecular
sieves (5 beads each). The solution was pushed back and forth through the synthesis
column for 2 min and left to react 4 hours at 30°C.
The deprotecting solution was removed by deconnecting the syringes from the column
without moving the plunger. The column was rinsed with 1 mL of dry THF followed by a
gently 1 min flush with argon. Both columns were eluted with 2 mL of 25 mM TEAB in the
same 100 mL flask. Then the mixtures were evaporated under reduced pressure at 30°C
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maximum until the volumes were reduced to 0.3 mL. The residues were redissolved in water
(1.5 mL divided in three portions for flask rinse: 0.8 mL, 0.4 mL, 0.3 mL) and transferred to 2
mL Eppendorf-vials then lyophilized from water.
Analytical and purification of GpppRNAs (13, 16, 19) and 7mGpppRNAs (14, 17, 20)
The crude GpppRNAs (13, 14, 16, 17, 19, 20) were analyzed and purified on a Dionex U
3000 HPLC system monitored at 260 nm and 300 nm with the columns kept at 50°C in a
thermostated column compartment. Analytical IEX-HPLC was performed using a DNAPac
PA-200 column (4 X 250 mm, Dionex). GpppRNAs (13, 14, 16, 17, 19, 20) were eluted using
a 0%-50% or 0%-75% linear gradient of buffer B in buffer A, flow rates were 1.2 mL.min-1.
The crude mixtures were purified by semi-preparative HPLC with a 0%-30% or 0%-75%
linear gradient of buffer B in buffer A. The fractions containing the pure GpppRNAs were
pooled in a 100 mL round-bottomed flask and were concentrated to a volume of 0.5 mL
under reduced pressure with a bath at 30°C. Then the pure GpppRNAs were ready for the
desalting procedure.
Desalting procedure for pure GpppRNAs and 7mGpppRNAs
Next to evaporation, the purified materials were redissolved in TEAB 100 mM (6 mL divided
in three portions for flask rinse: 3 mL, 2 mL, 1mL) and were loaded on a Sep Pak C18
cartridge. Elution was performed with 10 mL of 100 mM TEAB, pH 7 then with 10 mL of 50%
CH3CN in 12.5 mM TEAB, pH 7. The second fraction containing the desired 5’-capped RNA
was collected in a 100 mL round-bottomed flask and was freeze-dried. The residue was
dissolved in 1.5 mL water (divided in 3 portions of 0.8 mL, 0.4 mL, 0.3 mL for flask rinse) and
transferred to a 2 mL Eppendorf-vial and lyophilized from water. Lyophilized GpppRNAs and 7mGppp-RNAs were stored at -20°C for several months without any degradation.
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Table: Synthesized 5’-GpppT6 and 5’-GpppRNAs
ON
Catalysts[a]
5’-sequence-3’
Molecular Calc. Found
Formula m/z[b] m/z[b]
1 ZnCl2 GpppT6 C70H93N17O57 P8 2267.35 2267.29
2 NiCl2 GpppT6 C70H93N17O57 P8 2267.35 2267.61
N. caerulescens extract
3 crude GpppT6 C70H93N17O57 P8 2267.35 2267.34
4 purified (f1) GpppT6 C70H93N17O57 P8 2267.35 -
5 purified (f3) [c] GpppT6 C70H93N17O57 P8 2267.35 2267.25
6 purified (f3) GpppT6 C70H93N17O57 P8 2267.35 2267.65
N. caerulescens + A. vulneraria extract
7 crude GpppT6 C70H93N17O57 P8 2267.35 2267.57
P. douarrei extract
8 crude GpppT6 C70H93N17O57 P8 2267.35 2267.36
9 purified[c] GpppT6 C70H93N17O57 P8 2267.35 2267.80
G. pruinosa extract
10 crude GpppT6 C70H93N17O57 P8 2267.35 2267.34
11 purified GpppT6 C70H93N17O57 P8 2267.35 2267.29
P. accuminata extract
12 crude GpppT6 C70H93N17O57 P8 2267.35 2267.19
13
ZnCl2
GpppAGUUGUUAGUCUUACUGG
C151H225N70O141 P20
6254.57
6254.86
14 ZnCl2 GpppAUAUUA C67H83N26O53 P8 2348.31 2348.55
15 ZnCl2 7mGpppAUAUUA
C68H85N26O53 P8 2362.34 2362.67
N. caerulescens extract
16 purified (f3) GpppAOMeGUUGUUAGUCUUACUGGA C192H239N75O145 P21 6597.80 6597.45
17 purified (f3) GpppAUAUUA C67H83N26O53 P8 2348.31 2348.15
18 purified (f3) [d] 7mGpppAUAUUA C68H85N26O53 P8 2362.34 2362.59
P. douarrei extract
19 purified GpppAOMeGUUGUUAGUCUUACUGGA C192H239N75O145 P21 6597.80 6597.03
20 purified 7mGpppAUAUUA C68H85N26O53 P8 2362.34 2362.36
21 purified GpppAUAUUA C67H83N26O53 P8 2348.31 2348.32
[a] [Zn]=[Ni]= 0.4µm [b] MALDI-TOF characterization in negative mode [c] The catalyst-GDP solution was not centrifuged, but final mixture was eluted on Sephadex [d] The catalyst was treated by the system Zn/dioxan to eliminate traces of HCl and to prevent depurination
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0,0 5,0 10,0 15,0 20,0 25,0
12,71 13,9515,80
16,94GpppT6
GppT6
Im-pT6pT6
ON 1
0,0 5,0 10,0 15,0 20,0 25,0
12,71 13,9515,80
16,94GpppT6
GppT6
Im-pT6pT6
ON 1
Retention Time [minutes]
Figure S1: IEX-HPLC analysis of crude ON 1: GpppT6
IEX-HPLC analysis conditions: Column Dionex DNA PAC PA-100 (4 x 250 mm), elution with a linear gradient of 0% to 30% 400 mM NaClO4 in Tris-HCl 25 mM pH 8 / ACN 95/5, in 20 min, Flow rate 1.5 mL.min-1, 260 nm.
700 1360 2020 2680 3340 4000Mass (m/z)
0
10
20
30
40
50
60
70
80
90
100
% In
ten
sity
[M-H]- 2267.29
1841.16
1993.721132.74
218653664.33
ON 1
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0
10
20
30
40
50
60
70
80
90
100
% In
ten
sity
[M-H]- 2267.29
1841.16
1993.721132.74
218653664.33
ON 1
Figure S2: MALDI-TOF MS analysis of crude ON 1: GpppT6
Calc. For [M-H]- 2267.35
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0,0 5,0 10,0 15,0 20,0 25,0
13,62 14,8716,63
18,35GpppT6
GppT6
Im-pT6pT6
ON 2
0,0 5,0 10,0 15,0 20,0 25,0
13,62 14,8716,63
18,35GpppT6
GppT6
Im-pT6pT6
ON 2
Retention Time [minutes]
Figure S3: IEX-HPLC analysis of crude ON 2: GpppT6
IEX-HPLC analysis conditions: Column Dionex DNA PAC PA-100 (4 x 250 mm), elution with a linear gradient of 0% to 30% 400 mM NaClO4 in Tris-HCl 25 mM pH 8 / ACN 95/5, in 20 min, Flow rate 1.5 mL.min-1, 260 nm.
700 1360 2020 2680 3340 4000Mass (m/z)
0
10
20
30
40
50
60
70
80
90
100
% I
nte
nsi
ty
1842.32
[M-H]- 2267.61
1890.752187.29
3665.56
ON 2
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0
10
20
30
40
50
60
70
80
90
100
% I
nte
nsi
ty
1842.32
[M-H]- 2267.61
1890.752187.29
3665.56
ON 2
Figure S4: MALDI-TOF MS analysis of crude ON 2: GpppT6
Calc. For [M-H]- 2267.35
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0,0 5,0 10,0 15,0 20,0 25,0
13,55
14,85
16,32
18,14GpppT6
GppT6
Im-pT6
pT6
ON 3
0,0 5,0 10,0 15,0 20,0 25,0
13,55
14,85
16,32
18,14GpppT6
GppT6
Im-pT6
pT6
ON 3
Retention Time [minutes]
Figure S5: IEX-HPLC analysis of crude ON 3: GpppT6
IEX-HPLC analysis conditions: Column Dionex DNA PAC PA-100 (4 x 250 mm), elution with a linear gradient of 0% to 30% 400 mM NaClO4 in Tris-HCl 25 mM pH 8 / ACN 95/5, in 20 min, Flow rate 1.5 mL.min-1, 260 nm.
700 1360 2020 2680 3340 4000Mass (m/z)
0
10
20
30
40
50
60
70
80
90
100
% I
nte
nsi
ty
1842.21
[M-H]- 2267.34
3666.551925.781762.48
ON 3
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0
10
20
30
40
50
60
70
80
90
100
% I
nte
nsi
ty
1842.21
[M-H]- 2267.34
3666.551925.781762.48
ON 3
Figure S6: MALDI-TOF MS analysis of crude ON 3: GpppT6
Calc. For [M-H]- 2267.35
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0,0 5,0 10,0 15,0 20,0 25,0
13,89
pT6
ON 4
0,0 5,0 10,0 15,0 20,0 25,0
13,89
pT6
ON 4
Retention Time [minutes]
Figure S7: IEX-HPLC analysis of crude ON 4: GpppT6
IEX-HPLC analysis conditions: Column Dionex DNA PAC PA-100 (4 x 250 mm), elution with a linear gradient of 0% to 30% 400 mM NaClO4 in Tris-HCl 25 mM pH 8 / ACN 95/5, in 20 min, Flow rate 1.5 mL.min-1, 260 nm.
700 1360 2020 2680 3340 4000Mass (m/z)
0
10
20
30
40
50
60
70
80
90
100
% I
nte
nsi
ty
1842.52
1762.22 3684.921995.33
ON 4
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0
10
20
30
40
50
60
70
80
90
100
% I
nte
nsi
ty
1842.52
1762.22 3684.921995.33
ON 4
Figure S8: MALDI-TOF MS analysis of crude ON 4: GpppT6
Calc. For [M-H]- 2267.35
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0,0 5,0 10,0 15,0 20,0 25,0
13,43
14,15
15,88
17,467
pT6
Im-pT6
GppT6
GpppT6
ON 5
0,0 5,0 10,0 15,0 20,0 25,0
13,43
14,15
15,88
17,467
pT6
Im-pT6
GppT6
GpppT6
ON 5
Retention Time [minutes]
Figure S9: IEX-HPLC analysis of crude ON 5: GpppT6
IEX-HPLC analysis conditions: Column Dionex DNA PAC PA-100 (4 x 250 mm), elution with a linear gradient of 0% to 30% 400 mM NaClO4 in Tris-HCl 25 mM pH 8 / ACN 95/5, in 20 min, Flow rate 1.5 mL.min-1, 260 nm.
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0
10
20
30
40
50
60
70
80
90
100
% I
nte
nsi
ty
[M-H]- 2267.25
1994.171841.60
1132.94
1457.592069.80
3667.61
1762.19 2186.91 3587.30
ON 5
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0
10
20
30
40
50
60
70
80
90
100
% I
nte
nsi
ty
[M-H]- 2267.25
1994.171841.60
1132.94
1457.592069.80
3667.61
1762.19 2186.91 3587.30
ON 5
Figure S10: MALDI-TOF MS analysis of crude ON 5: GpppT6
Calc. For [M-H]- 2267.35
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0,0 5,0 10,0 15,0 20,0 25,0
12,50 13,7415,27
16,61
pT6 Im-pT6GppT6
GpppT6
ON 6
0,0 5,0 10,0 15,0 20,0 25,0
12,50 13,7415,27
16,61
pT6 Im-pT6GppT6
GpppT6
ON 6
Retention Time [minutes]
Figure S11: IEX-HPLC analysis of crude ON 6: GpppT6
IEX-HPLC analysis conditions: Column Dionex DNA PAC PA-100 (4 x 250 mm), elution with a linear gradient of 0% to 30% 400 mM NaClO4 in Tris-HCl 25 mM pH 8 / ACN 95/5, in 20 min, Flow rate 1.5 mL.min-1, 260 nm.
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0
10
20
30
40
50
60
70
80
90
100
% I
nte
nsi
ty
[M-H]- 2267.65
1842.19
1994.83
1762.893667.522187.63
3587.41
ON 6
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0
10
20
30
40
50
60
70
80
90
100
% I
nte
nsi
ty
[M-H]- 2267.65
1842.19
1994.83
1762.893667.522187.63
3587.41
ON 6
Figure S12: MALDI-TOF MS analysis of crude ON 6: GpppT6
Calc. For [M-H]- 2267.35
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0,0 5,0 10,0 15,0 20,0 25,0
13,21
13,98
17,66GpppT6
pT6
Im-pT6ON 7
0,0 5,0 10,0 15,0 20,0 25,0
13,21
13,98
17,66GpppT6
pT6
Im-pT6ON 7
Retention Time [minutes]
Figure S13: IEX-HPLC analysis of crude ON 7: GpppT6
IEX-HPLC analysis conditions: Column Dionex DNA PAC PA-100 (4 x 250 mm), elution with a linear gradient of 0% to 30% 400 mM NaClO4 in Tris-HCl 25 mM pH 8 / ACN 95/5, in 20 min, Flow rate 1.5 mL.min-1, 260 nm.
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0
10
20
30
40
50
60
70
80
90
100
% I
nte
nsi
ty
1892.50
1825.05
1841.90
1994.71
3667.79[M-H]- 2267.57
ON 7
700 1360 2020 2680 3340 4000Mass (m/z)
0
10
20
30
40
50
60
70
80
90
100
% I
nte
nsi
ty
1892.50
1825.05
1841.90
1994.71
3667.79[M-H]- 2267.57
ON 7
Figure S14: MALDI-TOF MS analysis of crude ON 7: GpppT6
Calc. For [M-H]- 2267.35
Electronic Supplementary Material (ESI) for RSC AdvancesThis journal is © The Royal Society of Chemistry 2013
- S 15 -
0,0 5,0 10,0 15,0 20,0 25,0
13,07
14,44
16,00
17,54
pT6
Im-pT6
GppT6
GpppT6
ON 8
0,0 5,0 10,0 15,0 20,0 25,0
13,07
14,44
16,00
17,54
pT6
Im-pT6
GppT6
GpppT6
ON 8
Retention Time [minutes]
Figure S15: IEX-HPLC analysis of crude ON 8: GpppT6
IEX-HPLC analysis conditions: Column Dionex DNA PAC PA-100 (4 x 250 mm), elution with a linear gradient of 0% to 30% 400 mM NaClO4 in Tris-HCl 25 mM pH 8 / ACN 95/5, in 20 min, Flow rate 1.5 mL.min-1, 260 nm.
700 1360 2020 2680 3340 4000Mass (m/z)
0
10
20
30
40
50
60
70
80
90
100
% I
nte
nsi
ty
1841.10
[M-H]- 2267.36
1457.48 1762.033665.502187.07
ON 8
700 1360 2020 2680 3340 4000Mass (m/z)
0
10
20
30
40
50
60
70
80
90
100
% I
nte
nsi
ty
1841.10
[M-H]- 2267.36
1457.48 1762.033665.502187.07
ON 8
Figure S16: MALDI-TOF MS analysis of crude ON 8: GpppT6
Calc. For [M-H]- 2267.35
Electronic Supplementary Material (ESI) for RSC AdvancesThis journal is © The Royal Society of Chemistry 2013
- S 16 -
0,0 5,0 10,0 15,0 20,0 25,0
14,34 15,83
17,42GpppT6
GppT6Im-pT6
ON 9
0,0 5,0 10,0 15,0 20,0 25,0
14,34 15,83
17,42GpppT6
GppT6Im-pT6
ON 9
Retention Time [minutes]
Figure S17: IEX-HPLC analysis of crude ON 9: GpppT6
IEX-HPLC analysis conditions: Column Dionex DNA PAC PA-100 (4 x 250 mm), elution with a linear gradient of 0% to 30% 400 mM NaClO4 in Tris-HCl 25 mM pH 8 / ACN 95/5, in 20 min, Flow rate 1.5 mL.min-1, 260 nm.
700 1360 2020 2680 3340 4000Mass (m/z)
0
10
20
30
40
50
60
70
80
90
100
% I
nte
nsi
ty
[M-H]- 2267.80
2187.741842.30
1993.973667.84
1133.371762.20 3748.08
ON 9
700 1360 2020 2680 3340 4000Mass (m/z)
0
10
20
30
40
50
60
70
80
90
100
% I
nte
nsi
ty
[M-H]- 2267.80
2187.741842.30
1993.973667.84
1133.371762.20 3748.08
ON 9
Figure S18: MALDI-TOF MS analysis of crude ON 9: GpppT6
Calc. For [M-H]- 2267.35
Electronic Supplementary Material (ESI) for RSC AdvancesThis journal is © The Royal Society of Chemistry 2013
- S 17 -
0,0 5,0 10,0 15,0 20,0 25,0
13,68 14,95
18,29
GpppT6
Im-pT6pT6
ON 10
0,0 5,0 10,0 15,0 20,0 25,0
13,68 14,95
18,29
GpppT6
Im-pT6pT6
ON 10
Retention Time [minutes]
Figure S19: IEX-HPLC analysis of crude ON 10: GpppT6
IEX-HPLC analysis conditions: Column Dionex DNA PAC PA-100 (4 x 250 mm), elution with a linear gradient of 0% to 30% 400 mM NaClO4 in Tris-HCl 25 mM pH 8 / ACN 95/5, in 20 min, Flow rate 1.5 mL.min-1, 260 nm.
700 1360 2020 2680 3340 4000Mass (m/z)
0
10
20
30
40
50
60
70
80
90
100
% I
nte
nsi
ty
1841.81
1892.43
3667.92[M-H]- 2267.34
1994.311762.02
ON 10
700 1360 2020 2680 3340 4000Mass (m/z)
0
10
20
30
40
50
60
70
80
90
100
% I
nte
nsi
ty
1841.81
1892.43
3667.92[M-H]- 2267.34
1994.311762.02
ON 10
Figure S20: MALDI-TOF MS analysis of crude ON 10: GpppT6
Calc. For [M-H]- 2267.35
Electronic Supplementary Material (ESI) for RSC AdvancesThis journal is © The Royal Society of Chemistry 2013
- S 18 -
0,0 5,0 10,0 15,0 20,0 25,0
12,9814,26
15,80
17,33GpppT6
pT6
Im-pT6
GppT6
ON 11
0,0 5,0 10,0 15,0 20,0 25,0
12,9814,26
15,80
17,33GpppT6
pT6
Im-pT6
GppT6
ON 11
Retention Time [minutes]
Figure S21: IEX-HPLC analysis of crude ON 11: GpppT6
IEX-HPLC analysis conditions: Column Dionex DNA PAC PA-100 (4 x 250 mm), elution with a linear gradient of 0% to 30% 400 mM NaClO4 in Tris-HCl 25 mM pH 8 / ACN 95/5, in 20 min, Flow rate 1.5 mL.min-1, 260 nm.
700 1360 2020 2680 3340 4000Mass (m/z)
0
10
20
30
40
50
60
70
80
90
100
% I
nte
nsi
ty
[M-H]- 2267.291841.74
2186.97
3665.97
1993.42
1762.061920.82
ON 11
700 1360 2020 2680 3340 4000Mass (m/z)
0
10
20
30
40
50
60
70
80
90
100
% I
nte
nsi
ty
[M-H]- 2267.291841.74
2186.97
3665.97
1993.42
1762.061920.82
ON 11
Figure S22: MALDI-TOF MS analysis of crude ON 11: GpppT6
Calc. For [M-H]- 2267.35
Electronic Supplementary Material (ESI) for RSC AdvancesThis journal is © The Royal Society of Chemistry 2013
- S 19 -
0,0 5,0 10,0 15,0 20,0 25,0
12,43
13,73 15,24
16,78pT6
Im-pT6 GppT6
GpppT6
ON 12
0,0 5,0 10,0 15,0 20,0 25,0
12,43
13,73 15,24
16,78pT6
Im-pT6 GppT6
GpppT6
ON 12
Retention Time [minutes]
Figure S23: IEX-HPLC analysis of crude ON 12: GpppT6
IEX-HPLC analysis conditions: Column Dionex DNA PAC PA-100 (4 x 250 mm), elution with a linear gradient of 0% to 30% 400 mM NaClO4 in Tris-HCl 25 mM pH 8 / ACN 95/5, in 20 min, Flow rate 1.5 mL.min-1, 260 nm.
700 1360 2020 2680 3340 4000Mass (m/z)
0
10
20
30
40
50
60
70
80
90
100
% I
nte
nsi
ty
1841.09
[M-H]- 2267.19
1993.67
2187.11
3667.18
ON 12
700 1360 2020 2680 3340 4000Mass (m/z)
0
10
20
30
40
50
60
70
80
90
100
% I
nte
nsi
ty
1841.09
[M-H]- 2267.19
1993.67
2187.11
3667.18
ON 12
Figure S24: MALDI-TOF MS analysis of crude ON 12: GpppT6
Calc. For [M-H]- 2267.35
Electronic Supplementary Material (ESI) for RSC AdvancesThis journal is © The Royal Society of Chemistry 2013
- S 20 -
0,0 5,0 10,0 15,0 20,0 25,0
17,80
18,19
ON 13
0,0 5,0 10,0 15,0 20,0 25,0
17,80
18,19
ON 13
Retention Time [minutes]
Figure S25: IEX-HPLC analysis of crude ON 13: GpppAGU UGU UAG UCU UAC GUG
0,0 5,0 10,0 15,0 20,0 25,0
18,53
ON 13
0,0 5,0 10,0 15,0 20,0 25,0
18,53
ON 13
Retention Time [minutes]
Figure S26: IEX-HPLC analysis of pure ON 13: GpppAGU UGU UAG UCU UAC GUG
IEX-HPLC analysis conditions: Column Dionex DNA PAC PA-200 (4 x 250 mm), elution with a linear gradient of 0% to 75% 400 mM NaClO4 in Tris-HCl 25 mM pH 8 / ACN 95/5, in 20 min, 50°C, Flow rate 1.2 mL.min-1, 260 nm.
700 2560 4420 6280 8140 10000Mass (m/z)
0
10
20
30
40
50
60
70
80
90
100
% I
nte
nsi
ty
[M-H]- 6254.86
6397.885989.74
ON 13
700 2560 4420 6280 8140 10000Mass (m/z)
0
10
20
30
40
50
60
70
80
90
100
% I
nte
nsi
ty
[M-H]- 6254.86
6397.885989.74
ON 13
Figure S27: MALDI-TOF MS analysis of pure ON 13: GpppAGU UGU UAG UCU UAC GUG
Calc. For [M-H]- 6254.57
Electronic Supplementary Material (ESI) for RSC AdvancesThis journal is © The Royal Society of Chemistry 2013
- S 21 -
0,0 5,0 10,0 15,0 20,0 25,0
13,63
14,41ON 14
0,0 5,0 10,0 15,0 20,0 25,0
13,63
14,41ON 14
Retention Time [minutes]
Figure S28: IEX-HPLC analysis of crude ON 14: 7mGpppAUA UUA at =260 nm
IEX-HPLC analysis conditions: Column Dionex DNA PAC PA-200 (4 x 250 mm), elution with a linear gradient of 0% to 75% 400 mM NaClO4 in Tris-HCl 25 mM pH 8 / ACN 95/5, in 20 min, 50°C, Flow rate 1.2 mL.min-1, 260 nm.
0,0 5,0 10,0 15,0 20,0 25,0
14,41ON 14
0,0 5,0 10,0 15,0 20,0 25,0
14,41ON 14
Retention Time [minutes]
Figure S29: IEX-HPLC analysis of crude ON 14: 7mGpppAUA UUA at =300 nm
Electronic Supplementary Material (ESI) for RSC AdvancesThis journal is © The Royal Society of Chemistry 2013
- S 22 -
0,0 5,0 10,0 15,0 20,0 25,0
14,32ON 14
0,0 5,0 10,0 15,0 20,0 25,0
14,32ON 14
Retention Time [minutes]
Figure S30: IEX-HPLC analysis of pure ON 14: 7mGpppAUA UUA at 260 nm
0,0 5,0 10,0 15,0 20,0 25,0
14,32ON 14
0,0 5,0 10,0 15,0 20,0 25,0
14,32ON 14
Retention Time [minutes]
Figure S31: IEX-HPLC analysis of pure ON 14: 7mGpppAUA UUA at 300 nm
IEX-HPLC analysis conditions: Column Dionex DNA PAC PA-200 (4 x 250 mm), elution with a linear gradient of 0% to 75% 400 mM NaClO4 in Tris-HCl 25 mM pH 8 / ACN 95/5, in 20 min, 50°C, Flow rate 1.2 mL.min-1.
700 1360 2020 2680 3340 4000Mass (m/z)
0
10
20
30
40
50
60
70
80
90
100
% I
nte
nsi
ty
[M-H]- 2362.67
2386.19
2198.16
ON 14
700 1360 2020 2680 3340 4000Mass (m/z)
0
10
20
30
40
50
60
70
80
90
100
% I
nte
nsi
ty
[M-H]- 2362.67
2386.19
2198.16
ON 14
Figure S32: MALDI-TOF MS analysis of pure ON 14: 7mGpppAUA UUA
Calc. For [M-H]- 2362.34
Electronic Supplementary Material (ESI) for RSC AdvancesThis journal is © The Royal Society of Chemistry 2013
- S 23 -
0.0 5.0 10.0 15.0 20.0 25.0
GpppAUAUUA17.23
Im-pAUAUUA
pAUAUUA14.17
15.50
ON 15
0.0 5.0 10.0 15.0 20.0 25.0
GpppAUAUUA17.23
Im-pAUAUUA
pAUAUUA14.17
15.50
ON 15
Retention Time [minutes]
Figure S33: IEX-HPLC analysis of crude ON 15: GpppAUA UUA
0.0 5.0 10.0 15.0 20.0 25.0
17.52ON 15
0.0 5.0 10.0 15.0 20.0 25.0
17.52ON 15
Retention Time [minutes]
Figure S34: IEX-HPLC analysis of pure ON 15: GpppAUA UUA
IEX-HPLC analysis conditions: Column Dionex DNA PAC PA-100 (4 x 250 mm), elution with a linear gradient of 0% to 30% 400 mM NaClO4 in Tris-HCl 25 mM pH 8 / ACN 95/5, in 20 min, Flow rate 1.5 mL.min-1, 260 nm.
700 1260 1820 2380 2940 3500Mass (m/z)
0
10
20
30
40
50
60
70
80
90
100
% In
tensity
[M-H]- 2348.55
ON 15
700 1260 1820 2380 2940 3500Mass (m/z)
0
10
20
30
40
50
60
70
80
90
100
% In
tensity
[M-H]- 2348.55
ON 15
Figure S35: MALDI-TOF MS analysis of pure ON 15: GpppAUA UUA
Calc. For [M-H]- 2348.31
Electronic Supplementary Material (ESI) for RSC AdvancesThis journal is © The Royal Society of Chemistry 2013
- S 24 -
0,0 5,0 10,0 15,0 20,0 25,0
17,87
18,28ON 16
0,0 5,0 10,0 15,0 20,0 25,0
17,87
18,28ON 16
Retention Time [minutes]
Figure S36: IEX-HPLC analysis of crude ON 16: GpppAOMeGU UGU UAG UCU UAC GUG A
0,0 5,0 10,0 15,0 20,0 25,0
18,66ON 16
0,0 5,0 10,0 15,0 20,0 25,0
18,66ON 16
Retention Time [minutes]
Figure S37: IEX-HPLC analysis of pure ON 16: GpppAOMeGU UGU UAG UCU UAC GUG A
IEX-HPLC analysis conditions: Column Dionex DNA PAC PA-200 (4 x 250 mm), elution with a linear gradient of 0% to 75% 400 mM NaClO4 in Tris-HCl 25 mM pH 8 / ACN 95/5, in 20 min, 50°C, Flow rate 1.2 mL.min-1, 260 nm
800 2240 3680 5120 6560 8000Mass (m/z)0
10
20
30
40
50
60
70
80
90
100
% I
nte
nsi
ty
[M-H]- 6597.45
[M-2H]2-3300.00
6446.55[M-3H]3- 2200.08
ON 16
800 2240 3680 5120 6560 8000Mass (m/z)0
10
20
30
40
50
60
70
80
90
100
% I
nte
nsi
ty
[M-H]- 6597.45
[M-2H]2-3300.00
6446.55[M-3H]3- 2200.08
800 2240 3680 5120 6560 8000Mass (m/z)0
10
20
30
40
50
60
70
80
90
100
% I
nte
nsi
ty
[M-H]- 6597.45
[M-2H]2-3300.00
6446.55[M-3H]3- 2200.08
ON 16
Figure S38: MALDI-TOF MS analysis of pure ON 16: GpppAOMeGU UGU UAG UCU UAC GUG A
Calc. For [M-H]- 6597.80
Electronic Supplementary Material (ESI) for RSC AdvancesThis journal is © The Royal Society of Chemistry 2013
- S 25 -
0,0 5,0 10,0 15,0 20,0 25,0
13,7514,77
ON 17
0,0 5,0 10,0 15,0 20,0 25,0
13,7514,77
ON 17
Retention Time [minutes]
Figure S39: IEX-HPLC analysis of crude ON 17: 7mGpppAUA UUA at 260 nm
0,0 5,0 10,0 15,0 20,0 25,0
14,77ON 17
0,0 5,0 10,0 15,0 20,0 25,0
14,77ON 17
Retention Time [minutes]
Figure S40: IEX-HPLC analysis of crude ON 17: 7mGpppAUA UUA at 300 nm
IEX-HPLC analysis conditions: Column Dionex DNA PAC PA-200 (4 x 250 mm), elution with a linear gradient of 0% to 75% 400 mM NaClO4 in Tris-HCl 25 mM pH 8 / ACN 95/5, in 20 min, 50°C, Flow rate 1.2 mL.min-1.
Electronic Supplementary Material (ESI) for RSC AdvancesThis journal is © The Royal Society of Chemistry 2013
- S 26 -
0,0 5,0 10,0 15,0 20,0 25,0
14,75ON 17
0,0 5,0 10,0 15,0 20,0 25,0
14,75ON 17
Retention Time [minutes]
Figure S41: IEX-HPLC analysis of pure ON 17: 7mGpppAUA UUA at 260 nm
IEX-HPLC analysis conditions: Column Dionex DNA PAC PA-200 (4 x 250 mm), elution with a linear gradient of 0% to 75% 400 mM NaClO4 in Tris-HCl 25 mM pH 8 / ACN 95/5, in 20 min, 50°C, Flow rate 1.2 mL.min-1.
0,0 5,0 10,0 15,0 20,0 25,0
14,75ON 17
0,0 5,0 10,0 15,0 20,0 25,0
14,75ON 17
Retention Time [minutes]
Figure S42: IEX-HPLC analysis of pure ON 17: 7mGpppAUA UUA at 300 nm.
700 1360 2020 2680 3340 4000Mass (m/z)
0
10
20
30
40
50
60
70
80
90
100
% I
nte
nsi
ty
[M-H]- 2362.59
[M-2H]2- 1180.83
2198.06
ON 17
700 1360 2020 2680 3340 4000Mass (m/z)
0
10
20
30
40
50
60
70
80
90
100
% I
nte
nsi
ty
[M-H]- 2362.59
[M-2H]2- 1180.83
2198.06
ON 17
Figure S43: MALDI-TOF MS analysis of pure ON 17: 7mGpppAUA UUA.
Calc. For [M-H]- 2362.34
Electronic Supplementary Material (ESI) for RSC AdvancesThis journal is © The Royal Society of Chemistry 2013
- S 27 -
0,0 5,0 10,0 15,0 20,0 25,0
14,8617,64
ON 18
0,0 5,0 10,0 15,0 20,0 25,0
14,8617,64
ON 18
Retention Time [minutes]
Figure S44: IEX-HPLC analysis of crude ON 18: GpppAUA UUA
0,0 5,0 10,0 15,0 20,0 25,0
17,53
ON 18
0,0 5,0 10,0 15,0 20,0 25,0
17,53
ON 18
Retention Time [minutes]
Figure S45: IEX-HPLC analysis of pure ON 18: GpppAUA UUA
IEX-HPLC analysis conditions: Column Dionex DNA PAC PA-100 (4 x 250 mm), elution with a linear gradient of 0% to 30% 400 mM NaClO4 in Tris-HCl 25 mM pH 8 / ACN 95/5, in 20 min, Flow rate 1.5 mL.min-1, 260 nm.
700 1360 2020 2680 3340 4000.0Mass (m/z)
0
10
20
30
40
50
60
70
80
90
100
% I
nte
nsi
ty
[M-H]- 2348.15
[M-2H]2- 1173.29
ON 18
700 1360 2020 2680 3340 4000.0Mass (m/z)
0
10
20
30
40
50
60
70
80
90
100
% I
nte
nsi
ty
[M-H]- 2348.15
[M-2H]2- 1173.29
ON 18
Figure S46: MALDI-TOF MS analysis of pure ON 18: GpppAUA UUA
Calc. For [M-H]- 2348.31
Electronic Supplementary Material (ESI) for RSC AdvancesThis journal is © The Royal Society of Chemistry 2013
- S 28 -
0,0 5,0 10,0 15,0 20,0 25,0
18,26
18,55
19,01
19,36
19,79
20,18
ON 19
0,0 5,0 10,0 15,0 20,0 25,0
18,26
18,55
19,01
19,36
19,79
20,18
ON 19
Retention Time [minutes]
Figure S47: IEX-HPLC analysis of 1h30 NH4OH ON 19: GpppAOMeGU UGU UAG UCU UAC GUG A
0,0 5,0 10,0 15,0 20,0 25,0
17,86
18,46
ON 19
0,0 5,0 10,0 15,0 20,0 25,0
17,86
18,46
ON 19
Retention Time [minutes]
Figure S48: IEX-HPLC analysis of crude ON 19: GpppAOMeGU UGU UAG UCU UAC GUG A
Retention Time [minutes]
Figure S49: IEX-HPLC analysis of crude ON 19 (after NAP-10 G-25 Sephadex) :
GpppAOMeGU UGU UAG UCU UAC GUG A
0,0 5,0 10,0 15,0 20,0 25,0
17,98
18,53
ON 19
0,0 5,0 10,0 15,0 20,0 25,0
17,98
18,53
ON 19
Electronic Supplementary Material (ESI) for RSC AdvancesThis journal is © The Royal Society of Chemistry 2013
- S 29 -
0,0 5,0 10,0 15,0 20,0 25,0
18,97ON 19
0,0 5,0 10,0 15,0 20,0 25,0
18,97ON 19
Retention Time [minutes]
Figure S50: IEX-HPLC analysis of pure ON 19: GpppAOMeGU UGU UAG UCU UAC GUG A
IEX-HPLC analysis conditions: Column Dionex DNA PAC PA-200 (4 x 250 mm), elution with a linear gradient of 0% to 75% 400 mM NaClO4 in Tris-HCl 25 mM pH 8 / ACN 95/5, in 20 min, 50°C, Flow rate 1.2 mL.min-1, 260
nm
700 2160 3620 5080 6540 8000Mass (m/z)
0
10
20
30
40
50
60
70
80
90
100
% I
nte
nsi
ty
[M-H]- 6597.03
[M-2H]2- 3300.486465.80
3836.61 5905.17
ON 19
700 2160 3620 5080 6540 8000Mass (m/z)
0
10
20
30
40
50
60
70
80
90
100
% I
nte
nsi
ty
[M-H]- 6597.03
[M-2H]2- 3300.486465.80
3836.61 5905.17
ON 19
Figure S51: MALDI-TOF MS analysis of pure ON 19: GpppAOMeGU UGU UAG UCU UAC GUG A
Calc. For [M-H]- 6597.80
Electronic Supplementary Material (ESI) for RSC AdvancesThis journal is © The Royal Society of Chemistry 2013
- S 30 -
0,0 5,0 10,0 15,0 20,0 25,0
13,65
14,81
ON 20
0,0 5,0 10,0 15,0 20,0 25,0
13,65
14,81
ON 20
Retention Time [minutes]
Figure S52: IEX-HPLC analysis of crude ON 20: 7mGpppAUA UUA at 260 nm
IEX-HPLC analysis conditions: Column Dionex DNA PAC PA-200 (4 x 250 mm), elution with a linear gradient of 0% to 75% 400 mM NaClO4 in Tris-HCl 25 mM pH 8 / ACN 95/5, in 20 min, 50°C, Flow rate 1.2 mL.min-1.
0,0 5,0 10,0 15,0 20,0 25,0
14,81
ON 20
0,0 5,0 10,0 15,0 20,0 25,0
14,81
ON 20
Retention Time [minutes]
Figure S53: IEX-HPLC analysis of crude ON 20: 7mGpppAUA UUA at 300 nm
700 1360 2020 2680 3340 4000Mass (m/z)
0
10
20
30
40
50
60
70
80
90
100
% I
nte
nsi
ty
1844.32 [M-H]- 2362.361208.52
2435.69
2198.38 3847.61
ON 20
1923.86
1978.47
700 1360 2020 2680 3340 4000Mass (m/z)
0
10
20
30
40
50
60
70
80
90
100
% I
nte
nsi
ty
1844.32 [M-H]- 2362.361208.52
2435.69
2198.38 3847.61
1844.32 [M-H]- 2362.361208.52
2435.69
2198.38 3847.61
ON 20
1923.86
1978.47
Figure S54: MALDI-TOF MS analysis of crude ON 20: 7mGpppAUA UUA.
Calc. For [M-H]- 2362.34
Electronic Supplementary Material (ESI) for RSC AdvancesThis journal is © The Royal Society of Chemistry 2013
- S 31 -
0,0 5,0 10,0 15,0 20,0 25,0
13,69
14,96
17,31
ON 21
0,0 5,0 10,0 15,0 20,0 25,0
13,69
14,96
17,31
ON 21
Retention Time [minutes]
Figure S55: IEX-HPLC analysis of crude ON 21: GpppAUA UUA
0,0 5,0 10,0 15,0 20,0 25,0
17,487
ON 21
0,0 5,0 10,0 15,0 20,0 25,0
17,487
ON 21
Retention Time [minutes]
Figure S56: IEX-HPLC analysis of pure ON 21: GpppAUA UUA
IEX-HPLC analysis conditions: Column Dionex DNA PAC PA-100 (4 x 250 mm), elution with a linear gradient of 0% to 30% 400 mM NaClO4 in Tris-HCl 25 mM pH 8 / ACN 95/5, in 20 min, Flow rate 1.5 mL.min-1, 260 nm.
700 1360 2020 2680 3340 4000Mass (m/z)
0
10
20
30
40
50
60
70
80
90
100
% I
nte
nsi
ty
[M-H]- 2348.32
[M-2H]2- 1173.79
ON 21
700 1360 2020 2680 3340 4000Mass (m/z)
0
10
20
30
40
50
60
70
80
90
100
% I
nte
nsi
ty
[M-H]- 2348.32
[M-2H]2- 1173.79
ON 21
Figure S57: MALDI-TOF MS analysis of pure ON 21: GpppAUA UUA
Calc. For [M-H]- 2348.31
Electronic Supplementary Material (ESI) for RSC AdvancesThis journal is © The Royal Society of Chemistry 2013