An Unconventional Acid-Labile Nucleobase Protection Concept for Guanosine Phosphoramidites in RNA Solid-Phase Synthesis

Article abstract of DOI:10.1002/chem.201605056

We present an innovative O⁶-tert-butyl/N²-tert-butyloxycarbonyl protection concept for guanosine (G) phosphoramidites. This concept is advantageous for 2′-modified G building blocks because of very efficient synthetic access when compared with existing routes that usually employ O⁶-(4-nitrophenyl)ethyl/N²-acyl protection or that start from 2-aminoadenosine involving enzymatic transformation into guanosine later on in the synthetic path. The new phosphoramidites are fully compatible with 2′-O-tBDMS or TOM phosphoramidites in standard RNA solid-phase synthesis and deprotection, and provide excellent quality of tailored RNAs for the growing range of applications in RNA biophysics, biochemistry, and biology.

Full text of DOI:10.1002/chem.201605056

A Journal of
Accepted Article
Title: An unconventional acid-labile nucleobase protection concept for
guanosine phosphoramidites in RNA solid-phase synthesis
Authors: Lukas Jud and Ronald Micura
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An unconventional acid-labile nucleobase protection concept for
guanosine phosphoramidites in RNA solid-phase synthesis
Lukas Jud,[a] and Ronald Micura*[a]
Abstract: We present an innovative O6-tert-butyl/N2-tert-butyloxy-
carbonyl protection concept for guanosine (G) phosphoramidites. This
concept is advantageous for 2’-modified G building blocks because of
very efficient synthetic access when compared to existing routes that
usually employ O6-(4-nitrophenyl)ethyl/N2-acyl protection or that start
from 2-aminoadenosine involving enzymatic transformation into
guanosine later on in the synthetic path. The novel phosphoramidites
are fully compatible with 2’-O-tBDMS or TOM phosphoramidites in
standard RNA solid-phase synthesis and deprotection, and provide
excellent quality of tailored RNAs for the growing range of applications
in RNA biophysics, biochemistry, and biology.
be quite different.18,19 For instance, although ribose 2’-modified
pyrimidine nucleoside phosphoramidtes are well accessible, the
corresponding purine counterparts – in particular guanosine
derivatives – are much more challenging and time-consuming in
terms of synthesis. This is true for simple 2’-O-aminoalkyl
modified building blocks, and even more for 2’-SeCH3, 2’-SCF3 or
2’-N3 modifications, to name a few.
To resolve this unsatisfying situation we devised a novel
protecting group pattern for guanosine phosphoramidites that
employs O6-tert-butyl, N2(bis-[tert-butyloxycarbonyl]) (O6-tBu, N2-
Boc2) protection of the guanine nucleobase (Figure 1). At first
sight, this seems disadvantageous because orthogonality to the
5’-O-DMT group during iterative detritylation during automated
strand assembly gets lost. However, our hypothesis was that this
pattern should nevertheless work efficiently in solid-phase
synthesis for the following reasons (Figure 1): After
phosphoramidite coupling, deprotection of tBu/Boc from guanine
bases as a concomitant result of the detritylation step of the
subsequent coupling cycle is not expected to cause interference,
referring to an early study by Y. Hayakawa and coworkers on
protection group-free DNA synthesis. These authors investigated
2’-deoxynucleoside phosphoramidites without base protection
and demonstrated that in contrast to NH2-free 2’-deoxycytidine or
Introduction
Naturally occurring nucleic acid modifications such as methylation
or pseudouridylation have attracted much attention lately because
of their unquestionable though rather poorly understood roles in
epigenetics.1-3 Likewise, artificial modifications have become
increasingly important in order to manipulate nucleic acid
properties and to create chemical diversity that is needed for
applications in the life sciences.4 Examples are manifold and
include nucleobase-, ribose-, and backbone modifications that, for
instance, can improve pharmacokinetic properties of therapeutic
oligonucleotides.5,6 Other examples include isoptope- and dye-
labeled RNAs for structural and functional analysis using NMR
and fluorescence spectroscopy.7-9
2’-deoxyadenosine
amidites,
the
corresponding
2’-
deoxyguanosine amidites were efficiently coupled without
byproducts, yielding DNA oligos in high quality.20 However, the
major problem with their approach was the reduced solubility and
aggregation of the NH2-free 2’-deoxyguanosine building block.
Solid-phase synthesis using nucleoside phosphoramidites is the
method of choice to introduce a modification of interest in site-
specific manner into RNA. Continuously optimized over the last
decades, RNAs of up to 50 to 70 nucleotides are readily
accessible.10-12 The nucleoside building blocks require suitable
protection of nucleobase amino and ribose 2’ hydroxy groups.
While the former are usually acyl protected, silyl protection
(tBDMS or TOM)13,14 of the latter are most widely applied, but also
orthoesters (ACE),15 2-cyanoethoxymethyl (CEM),16 or
thiocarbamates (TC)17 represent powerful alternatives. For all five
approaches, the syntheses of the standard nucleoside
phosphoramidites (A, C, G, U) have been very well optimized and
are robust. With respect to modified nucleosides the situation can
[a]
Mag. L. Jud, Prof. Dr. R. Micura
Institute of Organic Chemistry, Center for Molecular Biosciences
Innsbruck (CMBI), University of Innsbruck, Innrain 80-82, 6020
Innsbruck, Austria
Figure 1. A novel protection pattern (tBu/Boc) for guanosine phosphoramidites
(top left) that is compatible with standard solid-phase RNA synthesis, is
introduced in this work. After strand assembly on solid phase, the RNA carries
all protecting groups except the tBu/Boc groups that have been cleaved during
iterative detritylation (top right). Subsequently, standard deprotection provides
the free RNA (bottom). The approach is advantageous over existing ones for
the preparation of RNA with site-specific, 2’-modified guanosines.
E-mail: ronald.micura@uibk.ac.at
Supporting information for this article is given via a link at the end of
the document.
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Encouraged by the assumption that O6-tBu-N2(Boc)2 guanosine
phosphoramidites should be very well soluble, and importantly,
under the foreseeable aspect that such a protection pattern would
shorten and/or make synthetic routes towards 2’-modified
guanosine phosphoramidites more convenient, we set out to
realize this concept.
Because 2’,3’-O-tBDMS equilibration is time consuming, we
demonstrated that faster access to large amounts of building
block 2 can be achieved directly from guanosine via masking the
3’- and 5’-OH groups with the di-tert-butylsilyl clamp,24 followed
by tBDMS protection of the 2’-OH group to give compound 5
(Scheme 2). Then, introduction of the O6-tBu N2(Boc)2 pattern
was conducted with di-tert-butyldicarbonate in triethylamine to
provided 6 after chromatographic purification (64%). Selective
cleavage of 3’,5’-O-di-tert-butylsilyl moiety using HF in pyridine
gave the corresponding O6-tBu, N2(Boc)2 and 2’-O-tBDMS
protected guanosine 7 in 93% yield. Finally, tritylation and
phosphitylation following standard procedures furnished building
block 2. Starting from guanosine, our route yields 2 in a 29%
overall yield in five steps with four chromatographic purifications;
in total 1.2 g of 2 was synthesized in the course of this study.
Results and Discussion
First, we conceived the synthesis of the unmodified guanosine
building blocks 2 and 4 (Scheme 1) with either 2’-O-tBDMS or 2’-
O-TOM protection. Those should then be tested and evaluated
with respect to their compatibility in standard RNA solid-phase
synthesis, before moving on to 2’-modified guanosines. Access to
the starting material, namely O6-tert-butyl-N2(Boc)2 guanosine 1
in large quanities, was straightforward using the procedure
originally introduced by Montesarchio and coworkers,21 involving
two steps from commercially available 2’,3’,5’-O-triacetyl
guanosine22 with one chromatographic purification in 74% overall
yield. Tritylation of compound 1 followed standard conditions in
76% yield. Subsequently, compound 1a was tBDMS-protected
according to Ogilvie23 with silver nitrate in rather low
regioselectivity (about 3:2). However, the overall yield for 1b was
increased to 55% using base-induced 2’,3’-O-tBDMS equilibration.
Finally, phosphitylation with 2-cyanoethyl-N,N-diisopropylchloro-
phosphoramidite under basic conditions in THF gave building
block 2 in 86% yield.
Scheme 2. Alternative route to tBu/Boc2 protected guanosine phosphoramidite
2 for RNA solid-phase synthesis. Reagents and conditions: a. 1.1 equiv di-tert-
butyl silylditriflate, 5.0 equiv 1H-imidazole, 0 °C, 30 min, then 4.8 equiv tert-
butyldimethyl chlorosilane, 60 °C,
2 h, 72% of 5; b. 8 equiv di-tert-
butyldicarbonate, 9 equiv triethylamine, 0.5 equiv 4-(dimethylamino)pyridine,
RT, 2 d, 64% of 6; c. 0.9 M HF in pyridine/CH2Cl2, plastic vessel, 0 °C, 2 h, 93%
of 7; d. 1.1 equiv DMT-Cl, 0.1 4-(dimethylamino)pyridine, in pyridine, overnight,
RT, 78% of 7a (identical to 1b); e. 3 equiv 2-cyanoethyl-N,N-diisopropyl-
chlorophosphoramidite, 10 equiv 2,4,6-trimethylpyridine, 0.7 equiv N-
methylimidazole, in tetrahydrofuran, 1.5 h, RT, 86% of 2.
Concerning the corresponding guanosine building blocks for the
2’-O-{[(triisopropylsilyl)oxy]methyl}(TOM) RNA approach,14 we
remembered that the original paper reported high regioselectivity
of the tomylation reaction (with a ratio of 12:1 in favor of the 2’-O
over 3’-O isomer)14 when the exocyclic N2 amino group of the
guanine moiety was protected with a single acyl group only. In
analogy, we therefore decided to first cleave one of the two N2
Boc groups of compound 1. This was achieved in robust manner
using aqueous NaOH solution in methanol (Scheme 1).
Compound 3 was obtained in 88% yield and subsequent tritylation
under standard conditions gave 3a in 78% yield. Then, tomylation
of the 2’-hydroxyl group was conducted via the corresponding
cyclic 2’,3’-O-di-tert-butylstannylidene derivative formed in situ in
the presence of ethyldiisopropylamine and tert-Bu2SnCl2 in 1,2-
dichloroethane at 50°C. Treatment with 1.3 equivalents of TOM-
Cl14 gave a mixture of 2’-O and 3’-O-alkylated regioisomers in a
favorable ratio of 4:1. The desired 2’-O-alkylated derivative 3b
was easily isolated in pure form by chromatography on silica gel
Scheme 1. Synthesis of guanosine phosphoramidites with tBu and Boc
nucleobase protection. Reagents and conditions: a. 1.1 equiv DMT-Cl, 0.1 equiv
4-(dimethylamino)pyridine, 0.5 equiv EtN(iPr)2, in pyridine, 4.5 h, RT, 76% of
1a; b. 1.4 equiv AgNO3, 2.5 equiv tert-butyldimethylsilyl chloride (tBDMS-Cl),
2.3 equiv pyridine in THF, RT, 6 h, 55% of 1b (after one round of 2’-O,3’-O
tBDMS equilibration in methanol/triethylamine 9/1, 10 min, RT, starting from the
pure, isolated 3’-OtBDMS isomer; for a detailed protocol see ref. 7); c. 3 equiv
2-cyanoethyl-N,N-diisopropylchlorophosphoramidite,
10
equiv
2,4,6-
trimethylpyridine, 0.7 equiv N-methylimidazole, in tetrahydrofuran, 1.5 h, RT,
86% of 2; d. 0.13 M NaOH, in CH3OH/H2O (75/1), RT, overnight, 88% of 3; e.
1.3 equiv DMT-Cl, 0.1 equiv 4-(dimethylamino)pyridine, 1.3 equiv EtN(iPr)2, in
pyridine, 16 h, RT, 78% of 3a; f. 1.1 eqiv tert-Bu2SnCl2, 3.5 equiv
ethyldiisopropylamine, in ClCH2CH2Cl, RT, 2 h, then 1.3 equiv TOM-Cl, 3.4
equiv ethyldiisopropylamine, 50 °C,
6 h, separation of 2’ isomer by
chromatography (2’ isomer: 62% of 3b, 3’ isomer: 15%); g. 3 equiv 2-
cyanoethyl-N,N-diisopropylchlorophosphoramidite, 7.6 equiv EtN(iPr)2, in
CH2Cl2, 3.5 h, RT, 83% of 4.
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as the first-eluting isomer in 62% yield. For comparison, direct
tomylation of the N2-Boc2 derivative 1a gave only 32% yield of 2’-
O-TOM isomer while 40% of 3’-O-TOM isomer was isolated.
Starting with compound 1, our route provides 4 in a 35% overall
yield in four steps with four chromatographic purifications; in total,
1.8 g of 4 was obtained in the course of this study.
ionization (LC–ESI) mass spectrometry (MS) (Figure 2). This
supports our alleged hypothesis that the tBu/Boc groups at
guanine bases are concomitantly cleaved during cleavage of the
trityl groups (dichloroacetic acid in dichloroethane) when the RNA
strand is assembled on solid phase, and that the deprotected
guanine nucleobases do not interfere further with successive
phosphoramidite coupling.
Table 1. Selection of RNAs synthesized using the tBu/Boc protected
guanosine phosphoramidites 2, 4, 10, and 13 (indicated by G*).
[c]
RNA sequence 5’ to 3’[a]
nt[b]
#
m.w. calc
[amu]
m.w. obs
[amu]
UUA G*CG
6
6
2
1875.19
1931.22
3810.36
6674.05
1874.85
1930.90
3810.17
6673.85
UG*G* G*G*U
2
2
2
CG*C G*AA UUC G*CG
12
21
UAU CUU AUU G*G*C AG*A G*AC
CUG
UUA G*CG
6
8
4
4
4
1875.19
2509.58
3513.21
1875.07
2509.35
3513.21
4255.59
6247.52
CG*A UCG* AU
AUC AG*G* UG*C AA
GCC GCC (2’-SCF3 G)*U GGU A
11
Figure 2. Selection of RNAs that were synthesized using tBu/Boc protected
amidites 2 or 4 (indicated in italic style) Anion exchange HPLC traces (top) of
(a) 6 nt single-stranded RNA, (b) 6 nt G-quadruplex-forming RNA, (c) 21 nt RNA,
and (d) 8 nt RNA and corresponding LC-ESI mass spectra (bottom) HPLC
conditions: Dionex DNAPac column (4x250 mm), 80 °C, 1 mL min-1, 0-60 %
buffer B in 45 min; buffer A: Tris-HCl (25 mM), urea (6 M), pH 8.0; buffer B: Tris-
HCl (25 mM), urea (6 M), NaClO4 (0.5 M), pH 8.0. For LC-ESI MS conditions,
see Supporting Information. The RNA 3’-terminal guanosines originated from
solid supports and carried standard acyl protection patterns (see SI).
13 10 4255.63
19 10 6247.88
UGG U(2’-SCF3 G)*A AUG AAG CCA
CAG G
UGC UCC UAG UAC GAG AGG ACC
G(2'-O-(CH2)3NH2 G)*A GUA
27 13 8799.46
8799.32
[a] In addition to G* amidites (tBu/Boc), 2’-O-tBDMS nucleoside
phosphoramidites were used for the first four sequences while 2’-O-TOM
nucleoside phosphoramidites were used for subsequent six RNAs; [b]
Number of nucleotides (nt); [c] Phosphoramidite compound number (#).
To assess the novel tBu/Boc protection concept in standard RNA
solid-phase synthesis, we prepared a series of RNAs of different
length, containing varying numbers of guanosines in the
sequence (Figure 2, Table 1). Building blocks 2 and 4 were
applied in combination with standard N-acylated 2’-O-TOM and/or
2’-O-tBDMS adenosine, cytidine and uridine phosphoramidites.
The oligos were assembled on standard polystyrene or controlled
pore glass (CPG) supports, using the standard implementations
of solid-phase synthesis cycles on commercial DNA/RNA
synthesizers. Coupling yields of building blocks 2 and 4 were
higher than 98% according to the trityl assay.
Cleavage from the solid support and deprotection of the RNA was
performed under standard conditions with methylamine in
ethanol/water, or alternatively, in methylamine/ammonia in water
followed by treatment with TBAF in THF; importantly, no
additional (acidic) deprotection step was applied. Salts were
removed by size-exclusion chromatography on a Sephadex G25
column. In general, the crude products gave a major product peak
in anion-exchange (AE) HPLC analysis (Figure 2). RNA oligomers
were purified by AE chromatography under strong denaturing
conditions (6 M urea, 80 °C). The molecular weights of the purified
RNAs were confirmed by liquid chromatography–electrospray
Our in-depth product analysis included minor peaks that eluted
slightly slower or faster than the desired major RNA peak. We
assigned these minor products to molecular weights that were
either 54 or 56 mass units higher than the desired RNA. The
former corresponds to the commonly occurring cyanoethyl-RNA
adducts (plus 54 amu)25 while the latter, most likely, refers to tert-
butyl RNA adducts (plus 56 amu). To support this hypothesis, we
changed the solvent of the detritylation solution from
dichloroethane to acetonitrile, which we expected to act as
carbocation scavenger,40 and indeed, the plus 56 amu byproduct
diminished below detection limit, or decreased significantly
(Supporting Figure S1).
A major driving force to develop the tBu/Boc guanine approach
was the foreseeable advantage to generate short and efficient
synthetic routes for 2’-modified guanosine building blocks of RNA
solid-phase synthesis. For instance, thus far, 2’-SeCH3 or 2’-N3
guanosine building block synthesis26,27 relied on N2-acetyl-O6-(4-
nitrophenyl)ethyl (NPE) or N2-dimethylformamidine-O6-NPE
functionalization to protect the guanine lactam moiety against
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electrophilic reagents. Introduction of the O6-NPE group was
accomplished under Mitsunobu conditions.28 Although optimized
over the years in our laboratory, we experienced unsatisfying
yields for this transformation. Additionally, extensive purification
protocols (to remove triphenylphosphine oxide) were required
rendering such a pathway time consuming and not very attractive.
We decided to test the O6-tBu-N2(Boc)2 pattern first for a 2’-SCF3
guanosine building block. Indeed, the O6-tBu N2(Boc)2 protected
guanosine derivative 829 can be readily transformed into the
phosphoramidite building block 10, and directly used in RNA
solid-phase synthesis (Scheme 3, Table 1, SI). Starting from
guanosine, the overall yield was 26 %. The present route is
therefore two steps shorter compared to the original synthesis
(14% overall yield)29 where we cleaved the O6tBu/N2Boc2 pattern
and replaced it by N2-[(dimethylamino)methylene] protection for
the final building block.29
availability of ADA is limited and back orders of several months
are not unusual representing an additional handicap. We also
consider our path superior to a more recent route that was
described by Herdewijn & coworkers for 2’-O-aminoethoxy(and
propoxy)methyl guanosine phosphoramidites because of ease-of-
handling.32
Scheme 4. Synthesis of 2’-(3-phthalimidopropoxymethyl) modified guanosine
phosphoramidite 13 carrying tBu/Boc nucleobase protection. Conditions: a. i.
1.05 equiv tBu2SnCl2, 3.3 equiv EtN(iPr)2, in CH2Cl2, RT, 1 h, ii. 1.2 equiv N-[3-
(Chloromethoxy)propyl]phthalimide 11, 5.2 equiv EtN(iPr)2, 50 °C, 6 h, 51% of
12; b. 3 equiv 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite, 8.9 equiv
EtN(iPr)2, in CH2Cl2, 3 h, RT, 84% of 13.
Scheme 3. Synthesis of 2’-SCF3 modified guanosine phosphoramidite 10
carrying tBu/Boc nucleobase protection. Conditions: a. 1.2 equiv DMT-Cl, 0.1
equiv 4-(dimethylamino)pyridine, 1.3 equiv EtN(iPr)2, in pyridine, 16 h, RT, 83%
of 9; b. 2.25 equiv 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite, 10
equiv EtN(CH3)2, in CH2Cl2, 3.5 h, RT, 78% of 10.
In addition, we demonstrate the convenience of the tBu/Boc
pattern for the synthesis of
a 2’-O-([3-phthalimidopropyl]-
oxymethyl) guanosine building block (13, Scheme 4, Table 1, SI).
Starting from compound 3a, alkylation of the 2’-OH via the cyclic
2’,3’-O-di-tert-butylstannylidene derivative formed in situ, followed
by treatment with ([3-phthalimidopropyl]oxymethyl)chloride 11
gave the 2’-O-([3-phthalimidopropyl]oxymethyl) guanosine
derivative 12 after chromatographic separation. Phosphitylation
under standard conditions furnished building block 13 in four
steps from compound 1 in 29% overall yield. Our path is therefore
significantly more efficient compared to a 10-step route that
required eight chromatographic purifications and gave
Scheme 5. Synthesis of 2’-azidoguanosine phosphodiester 18 carrying tBu/Boc
nucleobase protection. Conditions: a. i. 2.9 equiv trifluoromethanesulfonyl
chloride, 1.5 equiv 4-(dimethylamino)pyridine, 2.3 equiv triethylamine, in CH2Cl2,
0 °C, 40 min; ii. 5 equiv NaN3 in DMF, RT, overnight, 98% of 15 over two steps;
b. HF-pyridine, in CH2Cl2, 0 °C, 2 h, 79% of 16; c. 1.4 equiv DMT-Cl, 0.1 equiv
4-(dimethylamino)pyridine, in pyridine, RT, 20 h, 72% of 17; d. 5.4 equiv 1,2,4-
triazole, 4.9 equiv triethylamine, 2.4 equiv 2-chlorophenyldichlorophosphate,
3.9 equiv N-methylimidazole, in tetrahydrofuran, RT, 45 min, 78% of 18.
Compound 14 was synthesized in analogy to references [21,39].
appropriately
guanosine phosphoramidite in 6% overall yield.30 It is also more
convenient compared to 5-step route to 2’-O-(3-
protected
2’-O-(N-trifluoracetyl-2-aminoethyl)
a
phthalimidopropyl) guanosine phosphoramidite.31 Both these
routes start with 2-aminoadenosine and require enzymatic
transformation using adenosine deaminase (ADA) from calf
intestine. We conducted the 5-step route several times; in our
hands, the overall yield of 1% was very unsatisfying. Additionally,
We point out that we have a constant need for amino-modified
building blocks, such as derivative 13. They are required to
synthesize prefunctionalized RNA (containing NH2-, N3- and
alkyne tethers) for subsequent selective labeling with
fluorophores (Cy3, Cy5, Cy7, using NHS and Click chemistry)33,34
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4.58 (dd, J = 11.17 Hz, J = 5.70 Hz, 1H, H-C(2')); 4.92 (t, J = 6.00 Hz, 1H,
HO-C(5’)); 5.14 (d, J = 4.62 Hz, 1H, HO-C(3’)); 5.44 (d, J = 5.91 Hz, 1H,
HO-C(5’)); 5.86 (d, J = 6.00 Hz, 1H, H-C(1')); 8.32 (s, 1H, H-C(8)); 9.85 (s,
1H, NH) ppm. 13C-NMR (75 MHz, CDCl3): δ 28.02, 28.15 (2x OC(CH3)3);
61.48 (C(5‘)); 70.53 (C(3‘)); 73.49 (C(2‘)); 85.67 (C(4’)); 87.02 (C(1’));
140.40 (C(8)) ppm. ESI-MS: (m/z) [M+H]+ calculated for C19H30N5O7
440.22, found 440.10.
to generate high-performance biotinylated RNA probes for 2- and
3-color single-molecule FRET experiments to study RNA folding
and dynamics.35-37
As a final example to demonstrate the convenience of the new
protection pattern for 2’-modified
G building blocks, we
synthesized the 2’-deoxy-2’-azidoguanosine phosphodiester
derivative 18 (Scheme 5, SI). This derivative can be integrated
into a combined phosphotriester/phosphoramidite approach to
O6-tert-Butyl-N-(tert-butyloxycarbonyl)-5’-O-(4,4’-dimethoxytrityl)-
guanosine (3a). Compound 3 (1.0 g, 2.28 mmol) was coevaporated with
pyridine (6 ml) and subsequently dissolved in dry pyridine (7 ml). 4-
(Dimethylamino)pyridine (29 mg, 0.237 mmol) and N,N-diisopropyl
ethylamine (0.5 ml, 2.87 mmol) was added, followed by 4,4’-
dimethoxytriphenylmethyl chloride (880 mg, 2.62 mmol) in two portions
over a period of one hour. The solution was stirred for 13 hours before
more 4,4’-dimethoxytriphenylmethyl chloride (100 mg, 0.295 mmol) was
added and stirring was continued for three more hours. The reaction was
ended by the addition of methanol (0.5 ml) evaporated and subjected to
column chromatographic purification on SiO2 (1–6% CH3OH in
dichloromethane + 1% NEt3) yielding compound 3a as colorless foam
(1.32 g, 78%). TLC (6% CH3OH in dichloromethane + 1% NEt3) Rf = 0.74.
1H-NMR (300 MHz, CDCl3): δ 1.66 (s, 9H, boc); 1.83 (s, 9H, C(6)-O-
C(CH3)3); 3.29–3.33 (m, 2H, H1, H2-C(5’)); 3.86 (s, 6H, 2x OCH3) 4.51 (d,
J = 4.98 Hz, 1H, H-C(3')); 4.61 (triplettoid, 1H, H-C(4')); 5.03 (dd, J =
5.72 Hz, J = 8.12 Hz, 1H, H-C(2')); 6.07 (d, J = 6.40 Hz, 1H, H-C(1')); 6.83–
7.40 (m, 13H, HC(ar)); 7.72 (s, 1H, NH); 8.19 (s, 1H, HC(8)) ppm. 13C-
NMR (75 MHz, CDCl3): δ 28.23, 28.46 (2x O-C(CH3)3); 55.08 (2x OCH3);
63.81 (C(5‘)); 73.82 (C(3‘)); 76.57 (C(2‘)); 86.75 (C(4’)); 91.79 (C(1’));
113.07, 126.69–129.94 (C(ar)); 138.82 (C(8)) ppm. ESI-MS: (m/z) [M+H]+
calculated for C40H48N5O9 742.35, found 742.07.
synthesize 2’-azido RNA which is
bioconjugation and siRNA technologies.27
a
valuable tool for
Conclusions
We have introduced an unconventional, acid-labile protection
concept for G building blocks in RNA solid-phase synthesis. The
major strength of the O6-tBu N2(Boc)2 and O6-tBu N2HBoc pattern
is that synthetic routes towards 2’-modified G building blocks
become advantageous over existing concepts through handling
of well soluble derivatives and reducing the number of synthetic
steps. The novel tBu/Boc building blocks are fully compatible with
standard RNA synthesis and deprotection methods, making
RNAs with 2’-modified guanosines easily available. New
initiatives in RNA solid-phase synthesis – as the one described
here – are much needed to generate multiply labeled RNA for
biophysical applications and for diagnostic and biotechnological
approaches.
O6-tert-Butyl-N,N-bis(tert-butyloxycarbonyl)-3’,5’-O-di-tert-
butylsilanediyl-2’-O-tert-butyldimethylsilyl guanosine (6). Compound
538 (1.56 g, 2.90 mmol) was dissolved in acetonitrile (10 ml) and treated
with triethylamine (3.60 ml, 26.1 mmol), di-tert-butyldicarbonate (5.06 g,
23.2 mmol) and 4-(dimethylamino)pyridine (177 mg, 1.45 mmol) for 2 days.
The reaction was quenched with methanol and the volatiles were removed
under reduced pressure. The crude product was subjected to column
chromatographic purification on SiO2 (1–3% CH3OH in dichloromethane)
yielding compound 6 as yellow foam (1.46 g, 64%). TLC (6% CH3OH in
dichloromethane) Rf = 0.82. 1H-NMR (300 MHz, d6-DMSO): δ 0.07, 0.08
(2x s, 6H, Si(CH3)2); 0.87, 1.00, 1.06 (3x s, 27H, 3x tbu); 1.35 (s, 18H,
C(2)-N(Boc)2); 1.65 (s, 9H, C(6)-O-C(CH3)3); 3.93–4.01 (m, 2H, H-C(4'),
H-C(5')); 4.38 (dd, J = 7.76 Hz, J = 3.98 Hz, 1H, H-C(5')); 4.63 (d, J =
4.98 Hz, 1H, H-C(2')); (dd, J = 8.91 Hz, J = 5.04 Hz, 1H, H-C(3’)); 5.99 (s,
1H, H-C(1’)); 8.58 (s, 1H, H-C(8)) ppm. 13C-NMR (75 MHz, CDCl3): δ -4.22,
-4.87 (Si(CH3)2); 26.02–28.40 (6x C(CH3)3); 68.06 (C(5‘)); 75.01 (C(3‘));
75.70 (C(2‘)); 84.24 (C(4’)); 92.86 (C(1’)); 141.00 (C(8)) ppm. ESI-MS:
(m/z) [M+H]+ calculated for C38H68N5O9Si2 794.46, found 794.42.
Experimental Section
General. All chemicals were purchased from commercial suppliers and
used without further purification. All reactions were carried out under argon
atmosphere. Marchery-Nagel Polygram SIL G/UV254 pre-coated polyester
sheets (0.2 mm silica gel with fluorescent indicator) were used for thin
layer chromatography; the compounds were visualized at 254 nm. Flash
column chromatography was carried out on silica gel purchased from
Sigma-Aldrich (pore size 60 Å, 70–230 mesh, 63–200 µm). Triethylamine
(1%) was added to the solvents when packing silica gel columns. All
mixtures of liquids in this document are understood as v/v mixtures. Mass
spectrometry experiments were performed on a Finnigan LCQ Advantage
MAX ion trap instrument. 1H, 13C, 31P NMR spectra were recorded on a
Bruker DRX 300 MHz spectrometer; 19F NMR spectra were measured on
a Bruker Avance II+ 600 MHz spectrometer equipped with a TCI Prodigy
Cryo-Probe. The chemical shifts are referenced to the residual proton
signal of the deuterated solvents: CDCl3 (1H: 7.26 ppm, 13C: 77.1 ppm), d6-
DMSO (1H: 2.5 ppm, 13C: 39.5 ppm), 31P shifts are relative to external 85%
phosphoric acid, 19F shifts are relative to external CCl3F. 1H- and 13C-
assignments were based on COSY and HSQC experiments.
O6-tert-Butyl-N,N-bis(tert-butyloxycarbonyl)-2’-O-tert-
butyldimethylsilyl guanosine (7). Compound 6 (246 mg, 0.309 mmol)
was dissolved in dichloromethane (1.7 ml) in a plastic vessel, cooled in an
ice bath, and treated dropwise with HF/pyridine [prepared prior by diluting
47 µl (70% HF, 30% pyridine) with 280 µl pyridine in an ice cooled plastic
vessel]. The reaction was allowed to proceed at 0 °C for two hours before
it was ended and poured into a large excess of half saturated sodium
hydrogencarbonate solution. The mixture was extracted with
dichloromethane, dried over sodium sulfate, evaporated under reduced
pressure yielding compound 7 as white foam (189 mg, 93%) which was
used without further purification. TLC (6% CH3OH in dichloromethane) Rf
= 0.57. 1H-NMR (300 MHz, d6-DMSO): δ -0.17, 0.76 (2xs, 6H, 2x Si-
(CH3)2); 0.76 (s, 9H, Si-C(CH3)3); 1.38 (s, 18H, C(2)-N(Boc)2); 1.65 (s, 9H,
C(6)-O-C(CH3)3); 3.57, 3.71 (m, 2H, H1-, H2-C(5')); 3.99 (m, 1H, H-C(4'));
O6-tert-Butyl-N-(tert-butyloxycarbonyl)guanosine (3). Compound 121
(1.45 g, 2.69 mmol) was dissolved in methanol (75 ml) before an aqueous
sodium hydroxide solution (1.0 ml, 10 M) was added. The reaction mixture
was stirred overnight, evaporated to dryness and further dried under high
vacuum. The crude product was purified by column chromatographic
purification on SiO2 (6% CH3OH in dichloromethane) yielding compound 3
as colorless foam (1.05 g, 88%). TLC (6% CH3OH in dichloromethane) Rf
= 0.24. 1H-NMR (300 MHz, d6-DMSO): δ 1.48 (s, 9H, Boc); 1.67 (s, 9H,
C(6)-O-C(CH3)3); 3.50–3.68 (m, 2H, H1, H2-C(5’)); 3.93 (dd, J = 7.06 Hz,
J = 3.78 Hz, 1H, H-C(4')); 4.17 (dd, J = 7.94 Hz, J = 4.52 Hz, 1H, H-C(3'));
This article is protected by copyright. All rights reserved.

10.1002/chem.201605056
Chemistry - A European Journal
FULL PAPER
4.14 (dd, J = 4.92 Hz, J = 8.82 Hz, 1H, H-C(3')); 4.70 (triplettoid, 1H, H-
C(2')); 5.02 (m, 2H, HO-C(3’), HO-C(5’)); 5.92 (d, J = 5.34 Hz, 1H, H-C(1’));
8.63 (s, 1H, H-C(8)) ppm. 13C-NMR (75 MHz, d6-DMSO): δ -5.29, -5.06
(Si(CH3)2); 25.59 (SiC(CH3)3); 27.37 (N((CO)OC(CH3)3)2); 27.94 (O-
C(CH3)3); 61.03 (C(5‘)); 70.10 (C(3‘)); 75.33 (C(2‘)); 85.84 (C(4’)); 88.07
(C(1’)); 142.71 (C(8)) ppm. ESI-MS: (m/z) [M+H]+ calculated for
C30H52N5O9Si 654.35, found 654.27.
87.31, 88.40 (C(1’)); 113.43, 121.46–135.59 (C(ar)); 140.71 (C8) ppm. 31P-
NMR (121 MHz, CDCl3): δ 149.92, 151.17 ppm. ESI-MS: (m/z) [M+H]+
calculated for C60H87N7O12PSi 1157.45, found 1157.38.
O6-tert-Butyl-N-(tert-butyloxycarbonyl)-5’-O-(4,4’-dimethoxytrityl)-2’-
O-[(3-phthalimidopropoxy)methyl]guanosine (12). Compound 3a
(410 mg, 0.553 mmol) was dissolved in 1,2-dichloroethane (4 ml). N,N-
Diisopropylethylamine (320 µl, 1.84 mmol) and di-tert-butyltin dichloride
(176 mg, 0.579 mmol) were added, and the reaction mixture was stirred
for 2 hours at ambient temperature. After that time compound 11 (164 mg,
0.646 mmol) was added along with more N,N-diisopropylethylamine
(500 µl, 2.87 mmol). The reaction was allowed to proceed for one hour at
ambient temperature before it was heated to 50 °C for 6 hours. The
reaction was allowed to cool to ambient temperature, was quenched by
the addition of methanol, diluted with dichloromethane and washed with
aqueous half saturated sodium hydrogencarbonate solution. The
combined organic layers were dried over sodium sulfate, filtered and
evaporated under reduced pressure to yield, under high vacuum, the crude
product as red foam, which was subjected to column chromatographic
purification on SiO2 (toluene, hexane, ethylacetate (3/3/4) +1% NEt3) gave
pure 12 as colorless foam, and a fraction containing a mixture of 12 and
the corresponding 3’-O isomer. This fraction was again subjected to
column chromatographic purification on SiO2 (2% CH3OH in chloroform
+1% NEt3) to increase the total yield of 12 (270 mg, 51%). TLC (3%
CH3OH in dichloromethane) Rf = 0.46. 1H-NMR (300 MHz, CDCl3): δ 1.49
(s, 18H, C(2)-N(Boc)2); 1.74 (s, 9H, C(6)-O-C(CH3)3); 1.92 (m, 2H,
CH2CH2CH2); 2.91 (d, J = 5.76 Hz, 1H, HO-C(3’)); 3.25–3.65 (m, 6H,
CH2CH2CH2, H1-, H2-C(5’)); 3.76 (s, 6H, 2x OCH3); 4.19 (m, 1H, H-C(4'));
4.53 (dd, J = 5.41 Hz, J = 10.84 Hz, 1H, 1H, H-C(3')); 4.80 (m, 1H, H-C(2'));
4.86 (dd, J = 6.60 Hz, 2H, OCH2O); 6.09 (d, J = 3.69 Hz, 1H, H-C(1'));
6.77–7.42 (m, 13H, CH(ar)); 7.66, 7.78 (2x m, 4H, CH(ar)); 7.92 (s, 1H, H-
C(8)) ppm. 13C-NMR (75 MHz, CDCl3): δ 28.40, 28.67 (O-C(CH3)3,
N((CO)OC(CH3)3)2); 28.84 (CH2CH2CH2); 35.49 (CH2CH2CH2); 55.29 (2x
OCH3); 63.91 (C(5‘)); 65.87 (CH2CH2CH2); 76.00 (C(2’)); 78.04 (C(3‘));
84.87 (C(4’)); 91.17 (C(1’)); 96.01 (OCH2O); 123.28 127.87, 128.15,
130.09, 130.15 (C(ar)); 133.99 (C(8)) ppm. ESI-MS: (m/z) [M+H]+
calculated for C52H59N6O12 959.42, found 959.06.
O6-tert-Butyl-N,N-bis(tert-butyloxycarbonyl)-2’-O-tert-butyl dimethyl
silyl-5’-O-(4,4’-dimethoxy triphenylmethyl) guanosine (7a; identical to
1b, see SI). Compound 7 (1.0 g, 1.53 mmol) was coevaporated with dry
pyridine and dissolved in dry pyridine (5 ml). 4-(Dimethylamino)pyridine
(19 mg, 0.153 mmol) was added, and the reaction mixture was stirred for
30 min at ambient temperature before 4,4’-dimethoxytriphenylmethyl
chloride (570 mg, 1.68 mmol) was added in two portions over a period of
one hour. Stirring was continued overnight. After that time methanol
(700 µl) was added to end the reaction, immediately thereafter the solvents
were evaporated (note that extended reaction times would cause
isomerization of 2’/3’-O-tBDMS group!) and once coevaporated with
toluene. The crude product was subjected to column chromatographic
purification on SiO2 (0–2% CH3OH in dichloromethane, 1% NEt3) yielding
compound 7a (identical to 1b, SI) as yellow foam (1.13 g, 78%). TLC (3%
CH3OH in dichloromethane + 1% NEt3) Rf = 0.72. 1H-NMR (300 MHz,
CDCl3): δ 0.00, 0.03 (m, 6H, 2x C(2’)-O-Si-(CH3)2); 0.88 (s, 9H, C(2’)-O-
Si-C(CH3)3); 1.38 (s, 18H, C(2)-N(Boc)2); 1.71 (s, 9H, C(6)-O-C(CH3)3);
2.63 (d, J = 5.70 Hz, 1H, HO-C(3’)); 3.43, 3.48 (2x m, 2H, H1, H2-C(5’));
3.75 (s, 6H, 2x OCH3); 4.18 (m, 1H, H-C(4')); 4.28 (m, 1H, H-C(3')); 4.73
(triplettoid, 1H, H-C(2')); 6.05 (d, J = 4.14 Hz, 1H, H-C(1')); 6.80–7.44 (3x
m, 13H, H-C(ar)); 8.14 (s, 1H, H-C(8)) ppm. 13C-NMR (75 MHz, CDCl3): δ
-5.3, -4.57 (Si(CH3)2); 25.71 (SiC(CH3)3); 27.91 (N((CO)OC(CH3)3)2);
28.33 (O-C(CH3)3); 63.44 (C(5‘)); 71.28 (C(3‘)); 76.40 (C(2‘)); 83.93 (C(4’));
88.24 (C(1’)); 113.28, 127.00–130.06 (C(ar)); 140.42 (C(8)) ppm. ESI-MS:
(m/z) [M+H]+ calculated for C51H70N5O11Si 956.48, found 956.29.
O6-tert-butyl-N,N-bis(tert-butyloxycarbonyl)-2’-O-tert-
butyldimethylsilyl-5’-O-(4,4’-dimethoxytrityl)guanosine
3’-(2-cyanoethyl diisopropylphosphoramidite) (2). Compound 7a (or
1b, see SI) (500 mg, 0.523 mmol) was dissolved in absolute
tetrahydrofuran (2.5 ml) before 2,4,6-trimethylpyridine (694 µl, 5.23 mmol)
and N-methylimidazole (29 µl, 0.366 mmol) was added quickly.
Immediately thereafter, the resulting mixture was treated with 2-
cyanoethyl-N,N-diisopropylchlorophosphoramidite (372 mg, 1.57 mmol)
for 1.5 hours until TLC showed full conversion. The reaction was ended by
the addition of methanol (100 µl) and stirring was continued for 5 more
minutes. Within that time a thick white precipitate formed, which dissolved
when it was partitioned between ethylacetate and half saturated sodium
hydrogencarbonate solution. The organic layer was dried over sodium
sulfate and gave a yellow oil after evaporation. The crude product was
subjected to column chromatographic purification on SiO2 (7/3–5/5
hexane/ethylacetate, + 1% NEt3) yielding compound 2 as colorless foam
(521 mg, 86%). TLC (3% CH3OH in dichloromethane + 1% NEt3) Rf = 0.89.
1H-NMR (300 MHz, CDCl3): δ 0.00, 0.07 (m, 6H, 2x C(2’)-O-Si-(CH3)2);
0.84, 0.86 (m, 12H, N(CH(CH3)2)2); 0.91 (s, 9H, C(2’)-O-Si-C(CH3)3); 1.44,
1.45 (2x s, 18H, 2x C(2)-N(Boc)2); 1.78 (s, 9H, C(6)-O-C(CH3)3); 2.54 (2x
m, 2x 2H, OCH2CH2CN); 3.35–3.67 (m, 4H, H1-, H2-C(5'), N(CH(CH3)2)2);
3.83 (s, 6H, 2x OCH3); 4.05, 4.16 (2x m, 2H, OCH2CH2CN); 4.33–4.46 (m,
2H, H-C(3'), H-C(4')); 4.76, 4.85 (2x triplettoid, 1H, H-C(2')); 6.11, 6.14 (2x
d, J = 4.47 Hz, J = 5.10 Hz, 1H, H-C(1')); 6.88, -6.90, 7.20–7.43, 7.51 (3x
m, 13H, H-C(ar)); 8.24, 8.28 (2x s, 1H, H-C(8)) ppm. 13C-NMR (75 MHz,
CDCl3): δ -4.61 (2x C(2’)-O-Si-(CH3)2); 18.20, 20.22 (2x OCH2CH2CN);
O6-tert-butyl-N-(tert-butyloxycarbonyl)-5’-O-(4,4’-dimethoxytrityl)-2’-
O-[(3-phthalimidopropoxy)methyl]guanosine 3’-(2-cyanoethyl diiso-
propylphosphoramidite) (13). Compound 12 (159 mg, 0.166 mmol) was
dissolved in dichloromethane (3.5 ml). N,N-Diisopropylethylamine (258 µl,
1.48 mmol) and 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite
(118 mg, 0.498 mmol) were added and the reaction was allowed to
proceed at ambient temperature for 4 hours. After that time the reaction
was quenched by the addition of methanol and volatiles were evaporated.
The crude product was dissolved in a mixture of hexane/ethylacetate (6/4)
and filtered through a silica plug. The filtrate was evaporated and the crude
product was subjected to column chromatographic purification on SiO2
(ethylacetate/hexane 1/1–7/3) yielding compound 13 (162 mg, 84%) as
colorless foam. Alternative preparation:
A mixture of 12 and the
corresponding 2’-O isomer (80 mg, 1:1 ratio according to NMR analysis)
was dissolved in dichloromethane (2 ml) and treated with N,N-
diisopropylethylamine
(130 µl)
and
2-cyanoethyl-N,N-
diisopropylchlorophosphoramidite (90 mg, 0.380 mmol) for 3 hours at
ambient temperature. The reaction was ended by the addition of methanol
(300 µl) and stirring was continued for 5 minutes, before the reaction
mixture was diluted with dichloromethane and partitioned with half
saturated sodium hydrogencarbonate solution. The combined organic
layers were dried over sodium sulfate, filtered and evaporated. The crude
product was subjected to column chromatographic purification on SiO2
(ethylacetate/hexane 1/1–6/4), which yielded two well separated fractions.
The faster migrating fraction (TLC (ethylacetate/hexane 6/4) Rf = 0.78)
contained the two diastereomers of compound 13 as colorless foam
25.81
(C(2’)-O-Si-C(CH3)3);
25.93
(N(CH(CH3)2)2);
28.00
N((CO)OC(CH3)3)2); 28.45 (O-C(CH3)3); 49.90, 50.03 (2x C(5‘)); 55.25,
55.31 (2x OCH3); 56.64, 56.75 (OCH2CH2CN); 62.79, 63.11
(N(CH(CH3)2)2); 72.40, 72.57 (C(3‘)); 75.42, 75.46 (C(2‘)); 82.81 (C(4’));
This article is protected by copyright. All rights reserved.

10.1002/chem.201605056
Chemistry - A European Journal
FULL PAPER
(41 mg, 85%), as confirmed by 1H, 13C and 31P NMR analysis. The second
fraction (TLC (ethylacetate/hexane 6/4) Rf = 0.70) contained the two
diastereoisomers of O6-tert-butyl-N,N-bis(tert-butyloxycarbonyl)-5’-O-
(4,4’-dimethoxytrityl)-3’-O-[(3-phthalimidopropoxy)methyl]guanosine 2’-(2-
cyanoethyl diisopropylphosphoramidite). TLC (ethylacetate/hexane 6/4) Rf
= 0.78. 1H-NMR (300 MHz, CDCl3): δ 1.15, 1.17 (2x s, 12H, N(CH(CH3)2)2);
1.48 (s, 9H, C(2)-NH(boc)); 1.74 (s, 9H, C(6)-O-C(CH3)3); 1.86 (m, 2H,
fractions were lyophilized. Analysis of the quality of purified RNA was
performed by anion-exchange chromatography with same conditions as
for crude RNA; the molecular weight was confirmed by liquid
chromatography-electrospray ionization (LC-ESI) mass spectrometry.
Yields were determination by ultraviolet photometrical analysis of
oligonucleotide solutions.
CH2CH2CH2); 2.36, 2.60 (2x t,
J = 6.06 Hz, J = 6.03 Hz, 2H,
Mass spectrometry of RNA. All experiments were performed on a
Finnigan LCQ Advantage MAX ion trap instrumentation connected to an
Amersham Ettan micro LC system. RNA sequences were analysed in the
negative-ion mode with a potential of –4 kV applied to the spray needle.
LC: Sample (200 pmol RNA dissolved in 30 μl of 20 mM EDTA solution;
average injection volume: 30 μl); column (Waters XTerraMS, C18 2.5 μm;
1.0 x 50 mm) at 21 °C; flow rate: 30 μl min-1; eluant A: 8.6 mM triethylamine,
100 mM 1,1,1,3,3,3-hexafluoroisopropanol in H2O (pH 8.0); eluant B:
methanol; gradient: 0–100% B in A within 30 min; ultraviolet detection at
254 nm.
OCH2CH2CN); 3.40–3.67 (m, 10H, H1-, H2-C(5’), OCH2CH2CN,
N(CH(CH3)2)2, 2x CH2CH2CH2); 3.73, 3.76 (2x s, 6H, 2x OCH3); 3.85 (m,
2H, OCH2CH2CN); 4.11 (m, 2H, N(CH(CH3)2)2); 4.25, 4.34 (2x m, 1H, 2x
H-C(4')); 4.55 (m, 1H, H-C(3')); 4.67–4.74 (m, 3H, H-C(2'), OCH2O); 5.01,
5.10 (m, 2H, H-C(2’)); 6.02, 6.07 (2x d, J = 5.88 Hz, J = 4.77 Hz, 1H, H-
C(1')); 6.76–7.31 (m, 13H, H-C(arDMT)); 7.67, 7.79 (2x m, 4H, H-C(arphthal));
7.92, 7.94 (2x s, 1H, H-C(8)) ppm. 31P-NMR (121 MHz, CDCl3): δ 151.21,
151.61 ppm. ESI-MS: (m/z) [M+H]+ calculated for C61H76N8O13P 1159.53,
found 1159.26.
Solid-phase synthesis of RNA using tBu/Boc-protected guanosine
phosphoramidites. Standard phosphoramidite chemistry was applied for
RNA solid-phase synthesis using the novel O6-tert-butyl-N,N-bis(tert-
butyloxycarbonyl) or O6-tert-butyl-N-(tert-butyloxycarbonyl) protected
guanosine phosphoramidites (2, 4, 10, and 13) in combination with 2’-O-
tBDMS or 2’-O-TOM nucleoside phosphoramidite building blocks
(ChemGenes) and polystyrene support (GE Healthcare, Custom Primer
SupportTM, 80 μmol g-1; PS 200). All oligonucleotides were synthesized on
Acknowledgements
This work was supported by the Austrian Science Fund FWF
(projects I1040 and P27947). We thank Daniel Fellner, Elisabeth
Mairhofer, Sarah Klotz, and Sebastian Führer for synthetic
contributions.
a
ABI 392 Nucleic Acid Synthesizer following standard methods:
detritylation (80 s) with dichloroacetic acid/1,2-dichloroethane (4/96);
coupling (2.0 min) with phosphoramidites/acetonitrile (0.1 M x 130 μl) and
benzylthiotetrazole/acetonitrile (0.3 M x 360 μl); capping (3 x 0.4 min, Cap
A/Cap B = 1/1) with Cap A: 4-(dimethylamino)pyridine in acetonitrile (0.5
M) and Cap B: Ac2O/sym-collidine/acetonitrile (2/3/5); oxidation (1.0 min)
with I2 (20 mM) in tetrahydrofuran (THF)/pyridine/H2O (35/10/5). The
solutions of amidites and tetrazole, and acetonitrile were dried over
activated molecular sieves (4 Å) overnight.
Keywords: nucleoside • protecting groups • oligonucleotides •
RNA labeling • RNA modification
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Entry for the Table of Contents
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A novel tBu/Boc protection concept for
guanosine phosphoramidites is
Lukas Jud, Ronald Micura*
Page No. – Page No.
demonstrated that makes synthesis of
RNA with 2’-modified guanosines (G)
straightforward. RNA with selective G
labeling patterns are needed for
diverse applications in RNA
An unconventional acid-labile
nucleobase protection concept for
guanosine phosphoramidites for RNA
solid-phase synthesis
biophysics, biochemistry, and biology.
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