Practical synthesis of N-(di-n-butylamino)methylene-protected 2-aminopurine riboside phosphoramidite for RNA solid-phase synthesis

Article abstract of DOI:10.1007/s00706-019-02502-7

2-Aminopurine (Ap) is a fluorescent nucleobase analog that is frequently used as structure-sensitive reporter to study the chemical and biophysical properties of nucleic acids. In particular, thermodynamics and kinetics of RNA folding and RNA–ligand binding, as well as RNA catalytic activity are addressable by pursuing the Ap fluorescence signal in response to external stimuli. Site-specific incorporation of Ap into RNA is usually achieved by RNA solid-phase synthesis and requires appropriately functionalized Ap riboside building blocks. Here, we introduce a robust synthetic path toward a 2-aminopurine riboside phosphoramidite whose N² functionality is masked with the N-(di-n-butylamino)methylene group. This protection is considered advantageous over previously described N-(dimethylamino)methylene or acyl protection patterns needed for the fine-tuned deprotection conditions to achieve large synthetic RNAs. Graphic abstract: [Figure not available: see fulltext.].

Full text of DOI:10.1007/s00706-019-02502-7

Monatshefte für Chemie - Chemical Monthly
https://doi.org/10.1007/s00706-019-02502-7
ORIGINAL PAPER
Practical synthesis of N‑(di‑n‑butylamino)methylene‑protected
2‑aminopurine riboside phosphoramidite for RNA solid‑phase
synthesis
Eva Neuner1 · Ronald Micura1
Received: 9 July 2019 / Accepted: 9 September 2019
© The Author(s) 2019
Abstract
2-Aminopurine (Ap) is a fuorescent nucleobase analog that is frequently used as structure-sensitive reporter to study the
chemical and biophysical properties of nucleic acids. In particular, thermodynamics and kinetics of RNA folding and RNA–
ligand binding, as well as RNA catalytic activity are addressable by pursuing the Ap fuorescence signal in response to
external stimuli. Site-specifc incorporation of Ap into RNA is usually achieved by RNA solid-phase synthesis and requires
appropriately functionalized Ap riboside building blocks. Here, we introduce a robust synthetic path toward a 2-aminopurine
riboside phosphoramidite whose N2 functionality is masked with the N-(di-n-butylamino)methylene group. This protection
is considered advantageous over previously described N-(dimethylamino)methylene or acyl protection patterns needed for
the fne-tuned deprotection conditions to achieve large synthetic RNAs.
Graphic abstract
Keywords Nucleoside modifcation · Oligonucleotides · Fluorescence · 2ApFold · Ribozymes · Riboswitches
Introduction
RNA solid-phase synthesis based on appropriately function-
alized building blocks.
The nucleobase analog 2-aminopurine (Ap) is widely used
as minimally invasive fuorescent reporter in RNA for the
investigation of folding and ligand–RNA interactions [1–5].
Utilization of this label requires its site-specifc incorpora-
tion into the RNA of interest which is generally achieved by
Fox et al. [6] reported the frst synthesis of 2-aminopu-
rine riboside starting with the thiation of guanosine with
phosphorus pentasulfde followed by the reduction with
Raney nickel. Another synthetic route was published in the
same year by Schaefer et al. [7] utilizing the condensation
of chloromercuri-2-benzamidopurine with 2,3,5-tri-O-ben-
zoylribofuranosyl chloride followed by deprotection of the
hydroxyl functions. Alternatively, Nair et al. [8] introduced a
photochemical reduction of 6-chloro-2-aminopurine riboside
to the corresponding 2-aminopurine nucleoside.
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s00706-019-02502-7) contains
supplementary material, which is available to authorized users.
The frst incorporation of 2-aminopurine into DNA oli-
gomers, was reported by Eritja et al. [9] using the phos-
photriester method. The frst synthesis of a phosphor(III)
amidite of 2-aminopurine 2′-deoxyriboside and its
* Ronald Micura
ronald.micura@uibk.ac.at
1
Institute of Organic Chemistry, Leopold-Franzens
University, Innrain 80-82, Innsbruck, Austria
Vol.:(0123456789)
1 3

E. Neuner, R. Micura
incorporation into DNA was described by McLaughlin
et al. [10]. They obtained the nucleoside from the pre-
cursor of 6-hydrazino-2-aminopurine 2′-deoxyriboside in
the presence of AgO2. Several reports on the synthesis of
2-aminopurine 2′-deoxyriboside phosphoramidite and its
incorporation into DNA oligomers followed. Schmidt et al.
[11] reduced the protected 6-chloro-2-aminopurine ribo-
side with tri-n-butyltin hydride and azobis(isobutyronitril)
(AIBN) to obtain the desired 2-aminopurine 2′-deoxyribo-
side while Fujimoto et al. [12] prepared the Ap phosphora-
midite starting with the reduction of 6-thioguanosine using
Raney nickel and subsequent deoxygenation of the 2′-OH
via phenyl thiocarbonate formation and treatment with tri-
n-butyltin hydride and AIBN. Parel et al. [13] obtained
2-aminopurine 2′-deoxyriboside via glycosylation as had
been previously shown for glucopyranosyl-2-aminopurine
nucleosides by Garner et al. [14].
needed for the fne-tuned deprotection conditions to achieve
large synthetic RNAs.
Results and discussion
Our synthetic route to the functionalized 2-aminopurine
riboside phosphoramidite 6 starts with the reduction of the
commercially available 2-amino-6-chloropurine riboside
using Pearlman′s catalyst (Pd(OH)2/C) and ammonium
formate to yield compound 1 (Scheme 1). The exocyclic
2-amino function was selectively protected by treatment with
N,N-dibutylformamide dimethyl acetal (DBFDMA) [25–29]
producing nucleoside derivative 2. In the next step, the 5′ and
3′ hydroxyl groups were simultaneously protected by reac-
tion with di-tert-butylsilyl bis(trifuoromethanesulfonate)
((tBu)2Si(OTf)2) [30, 31], followed by silylation of the
2′-hydroxyl group with tert-butyldimethylsilyl chloride
(TBDMSCl) to give compound 3. The 5′ and 3′ hydroxyl
protection clamp was then selectively removed with a solu-
tion of HF in pyridine to yield compound 4. The function-
alization of the 5′ hydroxyl group with 4,4′-dimethoxytrityl
chloride was achieved under standard conditions to give
compound 5 in high yields. In the fnal step, the phospho-
ramidite 6 was generated by treatment with 2-cyanoethyl
N,N-diisopropylchlorophosphoramidite (CepCl) in the pres-
ence of 2,4,6-trimethylpyridine and N-methylimidazole in
THF, conditions that we applied to avoid migration of the
2′-O-TBDMS group during preparation of the ribonucleo-
side phosphoramidite [32]. Starting from 2-amino-6-chlo-
ropurine riboside, the target compound 6 was synthesized
in six steps, with fve chromatographic purifcations and an
overall yield of 33%.
The incorporation of 2-aminopurine into DNA was
soon complemented with reports on 2-aminopurine ribo-
side phosphoramidites for RNA synthesis by Doudna et al.
[15] as well as Santalucia et al. [16]. The key step in the
frst path involved the reduction of 6-thioguanosine with
Raney nickel whereas the second utilized the synthetic
conception from McLaughlin et al. [10]. The exocyclic
amine was protected with a benzoyl and isobutyryl group,
respectively, followed by 2′-O-tert-butyldimethylsilyl
(TBDMS) and 2′-O-tetrahydropyranyl protection, respec-
tively, to furnish the corresponding 2-aminopurine ribo-
side phosphoramidites. A similar synthesis was reported
by Tuschl et al. [17] for the investigation of hammerhead
ribozyme activity. Höbartner and co-workers [18] syn-
thesized the 2-aminopurine phosphoramidite containing
a 2′-O-TOM protecting group. Zagórowska et al. [19]
obtained the 2-aminopurine riboside phosphoramidite
by reduction of the triacetylated 6-chloro-2-aminopurine
riboside with hydrogen and palladium on carbon followed
by standard functionalization for TBDMS RNA chemistry.
Buchini et al. [20] and Peacock et al. [21] also reported
the hydrogenation of 6-chloro-2-aminopurine as key step
for the generation of 2′-O-aminoethyl or N2-alkylated
2-aminopurine riboside phosphoramidites. Koshkin [22]
employed a similar strategy using palladium hydroxide
on carbon and ammonium formate to hydrogenate the
6-chloro-2-aminopurine in locked nucleic acids (LNA)
nucleosides in high yields.
We note that the N-(di-n-butylamino)methylene-protected
2-aminopurine phosphoramidite 6 is coupled under standard
conditions for RNA solid-phase synthesis with yields com-
parable to the 2′-O-TBDMS standard nucleoside building
blocks. Oligoribonucleotides are deprotected under typical
deprotection conditions (e.g. a 1:1 mixture of 40% aqueous
methylamine and 30% aqueous ammonia (AMA) for 4 h at
room temperature or 45 min at 65 °C) (Supporting Fig. S1).
Also ultramild conditions, such as 4 h at RT with 0.05 M
potassium carbonate in methanol or 2 h at RT with ammo-
nium hydroxide can be applied, provided acetic anhydride
is replaced by phenoxyacetic anhydride for capping, and
phenoxyacetyl-protected phosphoramidite building blocks
are used in combination.
Here, we introduce a robust synthetic path for a 2-ami-
nopurine riboside phosphoramidite, starting from inex-
pensive 6-chloro-2-aminopurine riboside. In the target
compound, the N2 functionality is masked with the N-(di-
n-butylamino)methylene group [23, 24]. For 2-aminopurine
riboside building blocks, this protection is considered advan-
tageous over previously described N-(dimethylamino)meth-
ylene [15, 22] or acyl protection [10–13, 18, 19] patterns,
2-Aminopurine is a fuorescent isomer of adenine and
an important nucleobase modifcation for the elucidation
of structural and functional properties of nucleic acids. In
particular, several laboratories apply an approach termed
2-aminopurine-based RNA folding analysis (2ApFold) [2,
3, 33] which has been developed to provide insights into the
1 3

Practical synthesis of N-(di-n-butylamino)methylene-protected 2-aminopurine riboside p…
Scheme 1 Reaction conditions:
a
0.3 equiv Pd(OH)2/C, 10
(TBDMSCl) in DMF, 2 h, 60 °C, 85%; d 3.8 equiv HF-pyridine in
CH2Cl2, 0 °C, 2 h, 61%; e 1.2 equiv 4,4′-dimethoxytrityl chloride
(DMTrCl) in pyridine, 3 h, room temperature, 90%; f 0.6 equiv
1-methylimidazole, 7 equiv sym-collidine, 2.5 equiv 2-cyanoethyl
N,N-diisopropylchlorophosphoramidite (CepCl) in THF, room tem-
perature, 1.5 h, 84%
equiv ammonium formate in CH3OH:dioxane (1:1), 1 h, refux,
quantitative; b 3 equiv Bu2NCH(OCH3)2 in CH3OH, 2 d, room
temperature to 50 °C, 83%;
c i) 1.1 equiv di-tert-butylsilyl
bis(trifuoromethanesulfonate) ((tBu)2Si(OTf)2) in DMF, 30 min,
0 °C, ii) 5 equiv imidazole in DMF, 15 min at 0 °C, then 15 min at
room temperature, iii) 1.3 equiv tert-butyldimethylsilyl chloride
folding and folding dynamics of RNA. This approach relies
on synthetic RNAs with a single, strategically positioned
2-aminopurine which substitutes a nucleobase under mini-
mal steric interference and preservation of existing H-bond
and stacking patterns. Thereby, the selection of suitable Ap
positions is either based on the three-dimensional structure
(if available) or on SHAPE probing [3, 34, 35]. Hence, the
conformational rearrangements of the Ap-labeled RNA in
response to external stimuli (such as Mg2+ cations or low-
molecular weight compounds that bind RNA) are analyzed
by pursuing the Ap fuorescence intensity signal, reveal-
ing the underlying kinetic and thermodynamic parameters.
Beside the 2ApFold approach, a series of recent reports is
available that utilize 2-aminopurine fuorescence spectros-
copy to determine the cleavage kinetics of small nucleolytic
ribozymes [36–38].
features of the path are frst, hydrogenolysis of the 6-chlo-
ropurine derivative which we experienced advantageous in
terms of reproducibility over previously described routes
that involved reduction of 6-thioguanosine derivatives, and
second, N2 protection by the N-(di-n-butylamino)methyl-
ene group which allows the application of optimized, mild
deprotection conditions required for the preparation of large
synthetic RNA with more than 50 nucleotides.
Experimental
Reagents were purchased in the highest available quality
from commercial suppliers (Sigma-Aldrich, abcr, Carbo-
synth) and used without further purifcation. Moisture-sen-
sitive reactions were carried out under argon atmosphere. 1H
and 13C spectra were recorded on a Bruker DRX 400 MHz
spectrometer. Chemical shifts (δ) are reported relative to
tetramethylsilane (TMS) referenced to the residual pro-
ton signal of the deuterated solvent (DMSO-d6: 2.50 ppm
Conclusion
1
We have developed a convenient 6-step synthetic route
toward an amidine-protected 2-aminopurine riboside phos-
phoramidite for RNA solid-phase synthesis, starting from
inexpensive 2-amino-6-chloropurine riboside. The two key
for H spectra and 39.52 ppm for 13C spectra; CDCl3:
1
7.26 ppm for H spectra and 77.16 ppm for 13C spectra).
Signal assignments are based on 1H-1H-COSY and 1H-13C-
HSQC experiments. MS experiments were performed on
1 3

E. Neuner, R. Micura
a Thermo Scientifc Q Exactive Orbitrap with an electro-
spray ion source in the positive mode. Reaction control was
performed via analytical thin-layer chromatography (TLC,
Macherey–Nagel) with fuorescent indicator. Column chro-
matography was carried out on silica gel 60 (70–230 mesh).
vacuum, and dissolved in 2 cm3 dry N,N-dimethylformamide
in an ice bath. Di-tert-butylsilyl bis(trifuoromethanesulfonate)
(590 mg, 1.34 mmol) was added dropwise over a period of
15 min and the reaction mixture was stirred at 0 °C for 30 min.
Imidazole (419 mg, 6.15 mmol) was added and the mixture was
stirred for 15 min at 0 °C and for 15 min at room temperature.
Then, 241 mg tert-butyldimethylsiliyl chloride (1.59 mmol)
was added and the solution was stirred at 60 °C for another 2 h.
The mixture was diluted with dichloromethane, washed with
brine, dried over sodium sulfate, and evaporated. The crude
product was purifed by column chromatography on silica gel
(methanol:dichloromethane 0:100–2:98) to yield 695 mg (85%)
of 3 as white foam. TLC (methanol:dichloromethane 5:95):
Rf=0.46; HR-ESI–MS: m/z calculated for [C33H61N6O4Si2]+
([M+H]+): 661.4287, found 661.4269; 1H NMR (400 MHz,
CDCl3): δ=0.14–0.17 (d, 6H, 2× H2C(TBDMS)), 0.93 (9H,
H2C(TBDMS)), 1.04–1.06 (24H, 8× H3C), 1.32–1.42 (m,
4H, 2× H2C), 1.60–1.68 (m, 4H, 2× H2C), 3.30–3.34 (t, 2H,
H2CN), 3.58–3.63 (t, 2H, H2CN), 4.01–4.06 (m, 1H, HbC(5′)),
4.19–4.30 (m, 2H, HC(3′), HC(4′)), 4.49–4.52 (m, 1H, HaC(5′)),
4.55 (d, 1H, HC(2′)), 6.06 (1H, HC(1′)), 7.86 (1H, HC), 8.65
(1H, HC(8)), 8.90 (1H, HC(6)) ppm; 13C NMR (100 MHz,
CDCl3): δ=−4.78 (C(TBDMS)), −4.15 (C(TBDMS)), 13.87
(C(TBDMS)), 14.07 (C(TBDMS)), 19.98 (C(nbf)), 26.04
(C(TBDMS)), 27.15 (Si(C(CH3)3)2), 27.43 (C(nbf)), 29.36
(C(nbf)), 31.37 (C(nbf)), 45.46 (C(nbf)), 51.99 (C(nbf)), 68.00
(C(5′)), 74.52 (C(4′)), 75.72 (C(3′)), 76.47 (C(2′)), 91.41 (C(1′)),
140.96 (C(nbf)), 150.01 (C(6)), 157.92 (C(8)) ppm.
2‑Amino‑9‑(β‑d‑ribofuranosyl)purine (1, C10H13N5O4) 2-Amino-
6-chloropurine riboside (877 mg, 2.91 mmol) was dissolved in
15 cm3 methanol:dioxane (1:1) and 612 mg palladium hydrox-
ide on carbon (20 wt% loading, 0.872 mmol) and 1835 mg
ammonium formate (29.1 mmol) were added. The mixture
was refuxed for 1 h, cooled to room temperature, and fltered
through a Celite pad. The solvents were evaporated and the
product was dried under high vacuum. No further purifca-
tion was performed and a white solid was obtained in quan-
titative yield. HR-ESI–MS: m/z calculated for [C10H14N5O4]+
([M+H]+): 268.1040, found 268.1025; 1H NMR (400 MHz,
DMSO-d6): δ=3.51–3.56 (m, 1H, HbC(5′)), 3.60–3.66 (m,
1H, HaC(5′)), 3.89–3.92 (m, 1H, HC(4′)), 4.11–4.14 (m, 1H,
HC(3′)), 4.48–4.53 (m, 1H, HC(2′)), 5.06 (t, 1H, HO(5′)), 5.16
(d, 1H, HO(3′)), 5.44 (d, 1H, HO(2′)), 5.83 (d, 1H, HC(1′)),
6.55 (2H, H2N(2)), 8.30 (1H, HC(8)), 8.59 (1H, HC(6)) ppm;
13C NMR (100 MHz, DMSO-d6): δ=61.83 (C(5′)), 70.87
(C(3′)), 73.90 (C(2′)), 85.78 (C(4′)), 86.66 (C(1′)), 141.27
(C(8)), 149.74 (C(8)) ppm.
2‑[N‑(Di‑n‑butylamino)methylene]amino‑9‑(β‑d‑ribofuranosyl)‑
purine (2, C19H30N6O4) Compound 1 (770 mg, 2.88 mmol) was
dissolved in 5 cm3 methanol and 1758 mg N,N-dibutylforma-
mide dimethyl acetal (8.64 mmol) was added dropwise. The
solution was stirred at room temperature for 2 days and then
heated to 50 °C for 4 h. The solvents were evaporated and the
crude product was purifed by column chromatography on silica
gel (methanol:dichloromethane 1:99–10:90) to yield 969 mg
(83%) of 2 as white foam. TLC (methanol:dichloromethane
5:95): Rf=0.25; HR-ESI–MS: m/z calculated for [C19H31N6O4]+
([M+H]+): 407.2401, found 407.2387; 1H NMR (400 MHz,
CDCl3): δ=0.83–0.94 (m, 6H, 2× H3C), 1.29–1.34 (m, 4 H,
2× H2C), 1.55–1.63 (m, 4H, 2× H2C), 3.27–3.30 (m, 2H,
H2CN), 3.34–3.56 (m, 2H, H2CN), 3.67–3.71 (m, 1H, HbC(5′)),
3.85–3.88 (m, 1H, HaC(5′)), 4.23 (1H, HC(4′)), 4.41 (d, 1H,
HC(3′)), 4.99–5.02 (m, 1H, HC(2′)), 5.80 (d, 1H, HC(1′)), 7.88
(1H, HC), 8.42 (1H, HC(8)), 8.59 (1H, HC(6)) ppm; 13C NMR
(100 MHz, CDCl3): δ=13.87 (C(nbf)), 19.82 (C(nbf)), 29.15
(C(nbf)), 31.13 (C(nbf)), 45.60 (C(nbf)), 52.15 (C(nbf)), 62.96
(C(5′)), 72.34 (C(3′)), 73.16 (C(2′)), 87.06 (C(4′)), 90.46 (C(1′)),
143.38 (C(nbf)), 149.45 (C(6)), 158.26 (C(8)) ppm.
2‑[N‑(Di‑n‑butylamino)methylene]amino‑9‑(β‑d‑ribofuranosyl)‑
2′‑O‑(tert‑butyldimethylsilyl)purine (4, C22H44N6O4Si) Com-
pound 3 (695 mg, 1.05 mmol) was dissolved in 3 cm3 dichlo-
romethane in an ice bath. Hydrogen fuoride in pyridine (8 M,
0.1 cm3, 3.99 mmol) was diluted with 0.6 cm3 cold pyridine
and added dropwise to compound 3. The reaction mixture was
stirred at 0 °C for 2 h. Then, it was diluted with dichlorometh-
ane and saturated sodium bicarbonate was added. Stirring was
continued until no more gas evolution was observed, the organic
phase was washed twice more with saturated sodium bicarbo-
nate, dried over sodium sulfate, and evaporated. The crude
product was purifed by column chromatography on silica gel
(methanol:dichloromethane 0:100–3:97) to yield 332 mg (61%)
of 4 as white foam. TLC (methanol:dichloromethane 5:95):
Rf =0.42; HR-ESI–MS: m/z calculated for [C22H45N6O4Si]+
([M+H]+): 521.3266, found 521.3255; 1H NMR (400 MHz,
CDCl3): δ = − 0.19 (3H, H3C(TBDMS)), 0.00 (3H,
H3C(TBDMS)), 0.98 (9H, 3× H3C (TBDMS)), 1.11–1.15 (m,
6H, 2× H3C), 1.50–1.59 (m, 4H, 2× H2C), 1.76–1.82 (m, 4H,
2× H2C), 3.49–3.52 (m, 2H, H2CN), 3.66–3.89 (m, 2H, H2CN),
3.91–3.95 (m, 1H, HbC(5′)), 4.13–4.16 (m, 1H, HaC(5′)), 4.53
(2H, HC(3′), HC(4′)), 5.36–5.39 (t, 1H, HC(2′)), 5.91 (d, 1H,
HC(1′)), 7.99 (1H, HC), 8.73 (1H, HC(8)), 9.10 (1H, HC(6))
2‑[N‑(Di‑n‑butylamino)methylene]amino‑9‑(β‑d‑ribofuranosyl)‑
2′‑O‑(tert‑butyldimethylsilyl)‑3′,5′‑O‑(di‑tert‑butylsilylene)purine
(3, C33H60N6O4Si2) Compound 2 (495 mg, 1.23 mmol) was co-
evaporated three times with dry pyridine, dried under high
1 3

Practical synthesis of N-(di-n-butylamino)methylene-protected 2-aminopurine riboside p…
ppm; 13C NMR (100 MHz, CDCl3): δ=−5.30 (C(TBDMS)),
−5.19 (C(TBDMS)), 13.85 (C(TBDMS)), 13.99 (C(TBDMS)),
19.94 (C(nbf)), 20.26 (C(nbf)), 25.65 (C(TBDMS)), 29.30
(C(nbf)), 31.25 (C(nbf)), 45.48 (C(nbf)), 51.78 (C(nbf)), 63.40
(C(5′)), 73.02 (C(3′)), 74.06 (C(2′)), 87.61 (C(4′)), 91.08 (C(1′)),
143.17 (C(nbf)), 150.84 (C(6)), 158.01 (C(8)) ppm.
stirred at room temperature for 1.5 h. The reaction mixture
was diluted with dichloromethane, washed with saturated
sodium bicarbonate, and dried over sodium sulfate. The crude
product was purifed by column chromatography on silica gel
(ethyl acetate:cyclohexane 3:7–4:6) to yield 103 mg (84%)
of 6 as white foam. TLC (methanol:dichloromethane 3:97):
Rf =0.36 (both isomers); HR-ESI–MS: m/z calculated for
[C55H80N8O7PSi]+ ([M+H]+): 1023.5651, found 1023.5619;
1H NMR (400 MHz, CDCl3): δ=−0.163 (3H, H3C(TBDMS)),
0.00 (3H, H3C(TBDMS)), 0.77 (9H, H3C (TBDMS)), 0.92–
0.96 (m, 6H, (H3C)2CHN), 1.02 (3H, H3C), 1.16–1.20 (m, 9H,
(H3C)2CHN, H3C), 1.30–1.40 (m, 4H, 2× H2C), 1.57–1.68
(m, 4H, 2× H2C), 2.26–2.30 (m, 1H, HC(H3C)2N), 2.59–2.70
(m, 1H, HC(H3C)2N), 3.25–3.34 (m, 3H, H2CN, HaC(5′)),
3.44–3.48 (m, 1H, HbC(5′)), 3.34–3.63 (m, 6H, H2CN, H2CO,
H2CN), 3.78 (6H, 2× H3CO(DMT)), 4.30–4.38 (m, 2H, HC(3′),
HC(4′)), 4.73–4.78 (m, 1H, HC(2′)), 6.23–6.31 (dd, 1H, HC(1′)),
6.80–6.84 (m, 4H, HC(DMT)), 7.28–7.46 (m, 9H, HC(DMT)),
8.13–8.18 (1H, HC), 8.66 (1H, HC(8)), 8.88 (1H, HC(6)) ppm;
31P NMR (161 MHz, CDCl3): δ=149.22, 150.92 ppm.
2‑[N‑(Di‑n‑butylamino)methylene]amino‑9‑(β‑d‑ribofuranosyl)‑
5′‑O‑(4,4′‑dimethoxytrityl)‑2′‑O‑(tert‑butyldimethylsilyl)‑
purine (5, C46H62N6O6Si) Compound 4 (330 mg, 0.63 mmol)
was co-evaporated three times with pyridine, dried under
high vacuum for 1 h, and dissolved in 2 cm3 pyridine at
room temperature. 4,4′-Dimethoxytrityl chloride (258 mg,
0.78 mmol) was dried under high vacuum for 1 h prior to its
addition in 4 portions over 90 min to the solution. The reac-
tion mixture was stirred for 4 h at room temperature until the
starting material was fully consumed. Then, the solution was
diluted with dichloromethane, washed with 5% citric acid
and saturated sodium bicarbonate, dried over sodium sulfate,
and evaporated. The crude product was purifed by column
chromatography on silica gel (methanol:dichloromethane
0:100–2:98) to yield 465 mg (90%) of 5 as white foam. TLC
(methanol:dichloromethane 5:95): Rf =0.46; HR-ESI–MS:
m/z calculated for [C46H63N6O6Si]+ ([M+H]+): 823.4573,
found 823.4547; 1H NMR (400 MHz, CDCl3): δ=−0.15 (3H,
H3C(TBDMS)), 0.00 (3H, H3C(TBDMS)), 0.84 (9H, 3× H3C
(TBDMS)), 0.92–0.97 (m, 6H, 2× H3C), 1.31–1.40 (m, 4H, 2×
H2C), 1.56–1.66 (m, 4H, 2× H2C), 3.28–3.32 (m, 2H, H2CN),
3.37–3.52 (m, 2H, H2C(5′)), 3.59–3.65 (m, 2H, H2CN), 3.78
(6H, 2× H3CO(DMT)), 4.22-4.24 (m, 1H, HC(4′)), 4.31–4.34
(m, 1H, HC(3′)), 4.75 (t, 1H, HC(2′)), 6.25 (d, 1H, HC(1′)),
6.81–6.83 (m, 4H, HC(DMT)), 7.31–7.44 (m, 9H, HC(DMT)),
8.09 (1H, HC), 8.66 (1H, HC(8)), 8.89 (1H, HC(6)) ppm; 13C
NMR (100 MHz, CDCl3): δ=−5.05 (C(TBDMS)), −4.82
(C(TBDMS)), 13.88 (C(nbf)), 14.09 (C(nbf)), 19.94 (C(nbf)),
20.37 (C(nbf)), 25.73 (C(TBDMS)), 29.35 (C(nbf)), 31.30
(C(nbf)), 45.32 (C(nbf)), 51.82 (C(nbf)), 63.77 (C(5′)), 71.82
(C(3′)), 76.76 (C(2′)), 84.05 (C(4′)), 86.54 (C(1′)), 113.41
(C(DMT)), 127.19 (C(DMT)), 128.12 (C(DMT)), 128.24
(C(DMT)), 130.22 (C(DMT)), 141.04 (C(nbf)), 149.88 (C(6)),
158.09 (C(8)) ppm.
Acknowledgements Open access funding provided by Austrian Sci-
ence Fund (FWF). We thank Maximilian Himmelstoß and Thomas
Müller for mass spectrometric contributions. This work was supported
by the Austrian Science Fund FWF (Projects P27947 and P31691)
and the Austrian Research Promotion Agency FFG [West Austrian
BioNMR 858017].
Open Access This article is distributed under the terms of the Crea-
tive Commons Attribution 4.0 International License (http://creativeco
mmons.org/licenses/by/4.0/), which permits unrestricted use, distribu-
tion, and reproduction in any medium, provided you give appropriate
credit to the original author(s) and the source, provide a link to the
Creative Commons license, and indicate if changes were made.
References
1. Jean JM, Hall KB (2001) Proc Natl Acad Sci USA 98:37
2. Lang K, Rieder R, Micura R (2007) Nucl Acids Res 35:537
3. Souliere MF, Haller A, Rieder R, Micura R (2011) J Am Chem
Soc 133:16161
4. Kirk SR, Luedtke NW, Tor Y (2001) Bioorg Med Chem 9:2295
5. Sowers LC, Fazakerley GV, Eritja R, Kaplan BE, Goodman MF
(1986) Proc Natl Acad Sci USA 83:5434
2‑[N‑(Di‑n‑butylamino)methylene]amino‑9‑(β‑d‑ribofuranosyl)‑
5′‑O‑(4,4′‑dimethoxytrityl)‑2′‑O‑(tert‑butyldimethylsilyl)‑
purine 3′‑O‑2‑cyanoethyl‑N,N‑diisopropylphosphoramidite (6,
C55H79N8O7PSi) Compound 5 (100 mg, 0.12 mmol) was co-
evaporated three times with dry pyridine, three times with dry
toluene, and three times with dry tetrahydrofuran, and dried
under high vacuum for 1 h. It was dissolved in 1 cm3 dry tet-
rahydrofuran and 5.9 mg 1-methylimidazole (0.07 mmol) and
102 mg 2,4,6-collidine (0.84 mmol) were added subsequently.
Then, 71 mg 2-cyanoethyl N,N-diisopropylchlorophosphora-
midite (0.3 mmol) was added dropwise and the solution was
6. Fox JJ, Wempen I, Hampton A, Doerr IL (1958) J Am Chem Soc
80:1669
7. Schaefer JH, Thomas JH (1958) J Am Chem Soc 80:4896
8. Nair V, Young DA, Desilvia R (1987) J Org Chem 52:1344
9. Eritja R, Kaplan BE, Mhaskar D, Sowers LC, Petruska J, Good-
man MF (1986) Nucl Acids Res 14:5869
10. McLaughlin LW, Leong T, Benseler F, Piel N (1988) Nucl Acids
Res 16:5631
11. Schmidt S, Cech D (1995) Nucleosides Nucleotides 14:1445
12. Fujimoto J, Nuesca Z, Mazurek M, Sowers LC (1996) Nucl Acids
Res 24:754
13. Parel SP, Leumann CJ (2000) Helv Chim Acta 83:2514
1 3

E. Neuner, R. Micura
14. Garner P, Yoo JU, Sarabu R (1992) Tetrahedron 48:4259
15. Doudna JA, Szostak JW, Rich A, Usman N (1990) J Org Chem
55:5547
30. Serebryany V, Beigelman L (2002) Tetrahedron Lett 43:1983
31. Serebryany V, Beigelman L (2003) Nucleosides. Nucleotides Nucl
Acids 22:1007
16. Santalucia J, Kierzek R, Turner DH (1991) J Am Chem Soc
113:4313
32. Scaringe SA, Francklyn C, Usman N (1990) Nucl Acids Res
18:5433
17. Tuschl T, Ng MMP, Pieken W, Benseler F, Eckstein F (1993)
Biochemistry 32:11658
33. Haller A, Souliere MF, Micura R (2011) Acc Chem Res 44:1339
34. Souliere MF, Micura R (2014) Methods Mol Biol 1103:227
35. Souliere MF, Altman RB, Schwarz V, Haller A, Blanchard SC,
Micura R (2013) Proc Natl Acad Sci USA 110:3256
36. Ren A, Kosutic M, Rajashankar KR, Frener M, Santner T, West-
hof E, Micura R, Patel DJ (2014) Nat Commun 5:5534
37. Ren A, Vušurović N, Gebetsberger J, Gao P, Juen M, Kreutz C,
Micura R, Patel DJ (2016) Nat Chem Biol 12:702
18. Wachowius F, Höbartner C (2011) J Am Chem Soc 133:14888
19. Zagorowska I, Adamiak RW (1996) Biochimie 78:123
20. Buchini S, Leumann CJ (2006) Eur J Org Chem 2006:3152
21. Peacock H, Maydanovych O, Beal PA (2010) Org Lett 12:1044
22. Koshkin AA (2004) J Org Chem 69:3711
23. McBride LJ, Kierzek R, Beaucage SL, Caruthers MH (1986) J
Am Chem Soc 108:2040
38. Neuner S, Falschlunger C, Fuchs E, Himmelstoss M, Ren A, Patel
DJ, Micura R (2017) Angew Chem Int Ed 56:15954
24. Moyroud E, Biala E, Strazewski P (2000) Tetrahedron 56:1475
25. Falschlunger C, Micura R (2019) Monatsh Chem 150:795
26. Bredereck H, Simchen G, Rebsdat S, Kantlehner W, Horn P, Wahl
R, Hofmann H, Grieshaber P (1968) Chem Ber 101:41
27. Michel BY, Strazewski P (2009) Chemistry 15:6244
28. Froehler BC, Matteucci MD (1983) Nucl Acids Res 11:8031
29. Geiermann A-S, Micura R (2015) Curr Protoc Nucl Acid Chem
62(1):4–64
Publisher’s Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional afliations.
1 3