Acid-Stable Nucleobase Protection for a Strongly Pairing
Pyridone C-Nucleoside Suitable for Solid-Phase Synthesis of
Oligonucleotides
Juri Eyberg,[a] Daniela Göhringer,[a] Amila Salihovic,[a] and Clemens Richert*[a]

Oligonucleotides are indispensable tools in diagnostics, the-
rapeutic applications and molecular biology. The low base
pairing strength of thymine with adenine complicates their use.
Ethynylpyridone C-nucleosides are analogs of thymidine that
pair more strongly and with improved base selectivity, and
sequences containing these analogs show improved target
affinity and selectivity, but their routine use is hampered by
diminished yields of solid-phase syntheses with the known

building blocks. A partial loss of base protecting groups during
the acidic deblocking step of chain extension cycles was
identified as the cause of lower yields. Here we report the
synthesis of an improved phosphoramidite building block
featuring a pivaloyloxymethyl (POM) base protecting group.
This building block gives oligonucleotides containing the
strongly pairing ethynylmethylpyridone C-nucleoside in high
yield and purity via solid-phase synthesis.

Introduction

Synthetic oligodeoxynucleotides (ODNs) are invaluable in
diagnostics, therapeutics, molecular biology, bioorganic
chemistry, and nanostructuring. Their predictable binding to
complementary regions of target strands makes them useful as
hybridization probes, primers, antisense[1] or antigene agents,[2]

and building blocks for designed three-dimensional
assemblies,[3] to name just a few of their many applications.[4]

Solid-phase synthesis has made ODNs with canonical bases
readily available at very reasonable costs. Sequences with
modified bases are often more difficult to prepare, though,
making them interesting targets from a synthetic standpoint.

One motivation for developing syntheses of modified
oligonucleotides is to increase target affinity. The most weakly
binding base of the four canonical nucleobases of DNA is
thymine,[5] and A-rich target sequences can be difficult to bind
with oligonucleotides containing unmodified bases.[6] This has
prompted a quest for replacements of T that interact more
strongly with A than the natural deoxynucleoside.[7–9] In this
context, C-nucleosides have recently come into focus as
surrogates with increased affinity for adenine in target strands.
In particular, ethynylpyridones have shown potential to over-

come the poor target affinity and fidelity of thymine or
uracil.[10–14]

While several methods for setting up the C-nucleoside
framework are known,[15,16] the incorporation in oligonucleotides
usually relied on phosphoramidite or H-phosphonate-based
coupling[10] on controlled pore glass as solid support as the
predominant method. In automated syntheses, phosphorami-
dites are the preferred building blocks for chain assembly.[17]

This is why a phosphoramidite building block of the 6-ethynyl-
3-methylpyridone C-nucleoside, abbreviated “W”, which pairs
with adenine in target strands approximately as strongly as
deoxycytidine pairs with deoxyguanosine was developed.[18]

This pyridone building block (1, Scheme 1) has been
commercialized,[19] leading to more extensive use in the syn-
thesis of ODNs. While syntheses of strands containing one or
several W residues were successful, and duplex stabilization was
observed for each sequence tested,[18] side products were
detected in crudes in a follow-up study.[20] The side reaction
causing them was attributed to partial loss of pivaloyl (Piv)

[a] J. Eyberg, D. Göhringer, A. Salihovic, Prof. C. Richert
Institute of Organic Chemistry, University of Stuttgart
70569 Stuttgart, Germany
E-mail: lehrstuhl-2@oc.uni-stuttgart.de
https://chip.chemie.uni-stuttgart.de/
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/ejoc.202200611

This article belongs to a Joint Special Collection dedicated to Ulf Dieder-
ichsen.

© 2022 The Authors. European Journal of Organic Chemistry published by
Wiley-VCH GmbH. This is an open access article under the terms of the
Creative Commons Attribution Non-Commercial License, which permits use,
distribution and reproduction in any medium, provided the original work is
properly cited and is not used for commercial purposes.

Scheme 1. Removal of dimethoxytriyl (DMT) protecting groups with tri-
chloroacetic acid can lead to loss of pivaloyl (Piv) protecting groups of
ethynylpyridone C-nucleosides during automated DNA synthesis, resulting in
side products. Piv=pivaloyl, TCA= trichloroacetic acid, TIPS= triisopropylsil-
yl.

www.eurjoc.org

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http://orcid.org/0000-0002-6907-0205
https://chip.chemie.uni-stuttgart.de/
https://doi.org/10.1002/ejoc.202200611


groups during removal of dimethoxytrityl (DMT) 5’-protecting
group in the so-called ’deblock’ step of chain extension cycles,
which uses trichloroacetic acid in anhydrous dichloromethane.

The resulting free pyridone (2, Scheme 1) then coupled to
some of the the next incoming phosphoramidites, resulting in
detectable levels of branched products that were difficult to
hydrolyze fully to the desired linear strands.[20] Apparently,
protonation of the pyridine nitrogen makes the pivaloyl ester so
labile that even trace water in the deblock solution suffices to
induce hydrolysis. This motivated a search for a less acid labile
protecting group scheme for pyridone C-nucleosides. Here we
report the results of our study that resulted in a building block
with base protection that suppresses side reactions during ODN
synthesis.

Results and Discussion

The report of an industrial group[20] indicated that milder
Deblock reagents, such as dichloroacetic acid, can reduce the
loss of Piv groups, but deviating from the established standard
conditions of automated DNA synthesis was undesirable.
Instead, a more stable protecting group for the pyridone unit
was sought. Because protonation of the pyridinic nitrogen was
the most likely cause of lability under acidic conditions, N-
rather than O-protection was considered. However, it was
known from thymidine that even N3-Piv protected nucleosides
are unstable under acidic conditions.[21] Next, we tested other
acyl groups. Unfortunately, both acetyl and benzoyl groups
were unstable under the conditions of the Heck reaction
installing the pyridone. Derivatives with sterically more shielded
groups, such as the adamantoyl group employed for other non-
canonical nucleosides[22] or the mesitoyl group, were also
unstable or produced difficult-to-separate mixtures of N- and O-
protected products when introduced to the pyridone.
Carbonate and carbamate protecting groups, such as the
fluorenylmethoxycarbonyl (Fmoc)[23] or diphenyl carbamoyl
(DPC)[24] group also did not give the stability required to survive
all steps of the established synthetic route to the pyridone C-
nucleosides.[10,18]

Subsequently, ether protecting groups groups were tested.
The O-methyl derivative proved resistant to cleavage under
several conditions compatible with our nucleosides. The benzyl
protecting group is well established for the synthesis of C-
nucleosides,[10,25] and can be removed under Lewis acidic
conditions or by hydrogenolysis. Neither of the Lewis acids was
compatible with oligonucleotides, though, and hydrogenolysis
led to partial reduction of the ethynyl group. The more redox-
labile para-methoxybenzyl (PMB) group also could not be
readily removed after DNA synthesis. A diphenyl methyl ether,
similar to the one used for other nucleosides[26], was tested as a
less stable version of the benzyl ether, but attempts to obtain
the protected C-nucleoside in sufficient yield were unsuccessful.
Finally, an allyl ether was found to be incompatible with the
Heck reaction, due to the reactivity of its olefinic moiety.

As no appropriate ether protecting group was found, silyl
protecting groups were investigated. The widely used tert-

butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS) and
triisopropylsilyl (TIPS) ethers of the pyridone were either too
unstable or incompatible with at least one step of the synthetic
route. Next, a composite silyl protecting group with a two-step
deprotection mechanism was considered. One example for this
is the triisopropylsilyloxymethyl (TOM) protecting group used
for masking 2’-hydroxy groups during RNA syntheses. [27,28] Cross
reactivity with the silyl group of the glycal employed as educt
in the Heck reaction-based glycosylation was found to be
problematic, though.

A more suitable composite protecting group was then
identified that can be cleaved under basic conditions at the end
of automated DNA syntheses, concomitantly with the removal
of the acyl protecting groups on the nucleobases, the
cyanoethyl group protecting the phosphodiesters, and the
succinyl linkage to the solid support. This was the
pivaloyloxymethyl (POM) protecting group, comprised of a
formyl acetal and the pivaloyl group capping the distal hydroxy
functionality. The POM protecting group is known from the
phosphonate prodrug adefovir dipivoxyl,[29,30] as well as from
protected forms of uridine, thymidine,[31–34] imidazole C-nucleo-
sides, and pseudouridine.[35,36] In the latter cases, the nucleo-
bases are N-protected, though, and it was unclear what the
chemoselectivity would be for ethynylpyridones. Based on the
high O-reactivity of the uracil[10] and thymine analogs,[18] we
opted for phosphoramidite 3 as the preferred target molecule
for our syntheses.

Two routes to 3 are shown in Scheme 2. The most obvious
approach, introducing the POM group at the beginning of the
aglycone synthesis, was abandoned because the diazonium salt
intermediate used for the penultimate step in the elaboration
of the aglycone did not precipitate, and the crude reaction
mixtures gave significant side products upon conversion to the
iodide. Instead, the first route introduced the POM group on
the nucleoside level. For this, deoxynucleoside 4[18] was treated
with ammonia to obtain free pyridone C-nucleoside 5. Then,
the POM group was introduced using POM� Cl and carbonate in
DMF. Both O-alkylated 6 and N-protected 7 were isolated, at a
ratio of 3 :1, and a combined yield of 56%, as well as smaller
quantities of Piv-protected species resulting from transacylation
rather than alkylation. The structure of the N- and O-alkylated
pyridone C-nucleosides was confirmed by 2D NMR (see
Supporting Information). Both POM-protected pyridone C-
nucleosides, 6 and 7 were subjected to deblock conditions (3%
TCA in CH2Cl2) and the release of free nucleotide 5 was
monitored by UV absorption at the characteristic 323 nm
maximum of the free ethynylmethylpyridone.

Figure 1 shows the results of this stability study, together
with the kinetics for the corresponding O-pivaloyl protected 4.
Both POM-protected compounds passed the stability test,
whereas 4 showed the expected instability under these
conditions (no attempt to exclude residual water was made).
Among the two C-nucleosides with composite protecting
group, 7 appeared even more resistant to acidic cleavage,
remaining unchanged for 14 d, but 6 was chosen for further
elaboration to 3, via 5’-protected 8, as the more readily
accessible compound.

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The second route to 3 introduces the POM group earlier,
starting from 9,[18] and proceeding via free pyridone 10, which
was O-alkylated to POM-protected 11 in 82% yield, with just
9% of the N-protected counterpart formed. This higher chemo-
selectivity may be due to the bromine at the 2-position and its
steric and/or electronic effect. The POM-protected aglycone was
used in the Heck reaction with glycal 12, which was prepared as
described previously.[10] Thus, protected 13 was obtained in a
yield of 64% over 3 steps, including the cross coupling itself,
desilylation and stereoselective reduction to the 2’-deoxyribo-
nucleoside. Silyl enol ether 16 and ketone 17 were not purified,
as they are unstable, as described for similar molecules in the
literature.[10,18] The subsequent Sonogashira reaction gave 61%

yield, and the 5’-DMT protection to 8 and phosphitylation to 3
proceeded in 67% and 93% yield, respectively.

Phosphoramidite 3 was then used for the synthesis of two
different ODNs (Figure 2A). Oligonucleotide 14 is a sequence
previously prepared by the industrial group that reported the
branching problem.[20] The 5’-DMT protection was left in place
and deprotection in aqueous ammonia for 2 h at room temper-
ature was used to retain the TIPS groups, mirroring the
conditions of the literature work. Reversed-phase HPLC yielded
14 in 32%, and no later eluting peaks for branched oligonucleo-
tides were observed. This is in contrast to the corresponding
HPLC trace obtained with the Piv protecting group, which is
shown in ref. 20. The sequence of ODN 15 is that of a primer for
PCR tests detecting the SARS-COV-2 virus.[37] Here, solid-phase
chain assembly was followed by deprotection with ammonium
hydroxide for 16 h at 55 °C. Although about half of the TIPS
groups were removed during this step, no hydration of the
ethynyl residue, as described for similar ethynyl-substituted
bases,[38] was observed. After ammonia deprotection, the
remaining TIPS groups were removed with TBAF. The HPLC
trace of the crude showed no peaks for branching products
(Figure 2B), and 15 was obtained in 48% yield, i. e. the highest
yield published for an ODN containing an ethynylpyridone C-
nucleoside to date.

Conclusions

A broad screen of protecting groups identified the
pivaloyloxymethyl (POM) protecting group as an acid-stable
replacement for the pivaloyl group of the commercial building
block for the strongly pairing C-nucleoside W. The correspond-
ing phosphoramidite building block (3) was successfully
employed in the synthesis of oligonucleotides under standard
DNA synthesis conditions. The high-yielding synthesis of W-
containing strands opens the door to a routine use of the high-

Scheme 2. Synthesis of the POM protected phosphoramidite 3, starting from Piv protected aglycone 9 or C-nucleoside 4. Conditions: (a) NH4OH/MeCN, 99%;
(b) POM� Cl, K2CO3, DMF, 82%; (c) glycal 12, Pd(OAc)2, P(PhF5)3, Ag2CO3, CH3CN; (d) 3HF·NEt3, THF; (e) NaBH(OAc)3, CH3CN, 64% over 3 steps; (f)
triisopropylsilyacetylene, [Pd(PPh3)2Cl2], CuI, NEt3, DMF, 61%; (g) NH4OH/MeCN, 86%; (h) POM� Cl, K2CO3, DMF, 44%; (i) DMT� Cl, pyridine, 67%; (j)
(iPr2N)2P(OC2H4CN), DIPAT, CH3CN, 93%; DIPAT=diisopropylammonium tetrazolide, DMT=4,4’-dimethoxytrityl, Piv=pivaloyl, POM=pivaloyloxymethyl,
TIPS= triisopropylsilyl.

Figure 1. Stability of protected nucleosides to the Deblock solution of
automated DNA synthesis (3% TCA in dichloromethane), as monitored by
UV-absorbance at λmax=323 nm. A) reaction Scheme; B) Kinetics of formation
of free pyridone 5, starting from 82 μM solution of the respective nucleoside.
TCA= trichloroacetic acid.

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affinity replacement for thymidine and thus oligodeoxynucleo-
tides that can hybridize to their target sequences with high
affinity and fidelity.

Experimental Section
Reagents and solvents were purchased from Acros Organics (Geel,
Belgium), Alfa Aesar (Karlsruhe, Germany), Carbolution Chemicals
(Saarbrücken, Germany), Carbosynth (Compton, United Kingdom),
Thermo Fisher (Karlsruhe, Germany), Sigma-Aldrich (Schnelldorf,
Germany) or TCI Europe (Zwijndrecht, Belgium). All reactions were
performed under argon atmosphere. For thin layer chromatography
Macherey-Nagel ALUGRAM® Xtra SIL G/UV254 precoated silica gel
sheets were used, and the analytes were visualized by ultraviolet
light or staining with phosphomolybdatocerium(IV) sulfate solution
(25 g phosphomolybdic acid hydrate, 10 g cerium(IV) sulfate
tetrahydrate, 60 mL conc. sulfuric acid diluted with water to 1 L).
Column chromatography was carried out on silica from Macherey-
Nagel (Düren, Deutschland). The NMR spectra were measured on

Bruker Ascend 400, Avance 500 or Ascend 700 spectrometers . 1H-
NMR spectra were measured at 400, 500 or 700 MHz, 13C-NMR
spectra at 100, 125 or 175 MHz, and 31P-NMR-spectra were recorded
at 162 MHz. Chemical shifts are reported as δ values, relatively to
the chemical shifts of the solvent signal; coupling constants (J) are
given in Hz. Multiplicity is described as: s (singlet), br s (broad
singlet), d (doublet), t (triplet) or m (multiplet). High resolution
mass spectra were measured on a Bruker Daltonics microTOF� Q
mass spectrometer. MALDI-TOF mass spectra were measured in
linear, negative mode on a Bruker microflex spectrometer with the
matrix mixture 2,4,6-trihydroxyacetophenone (THAP, 0.3 M in
ethanol) and triammonium citrate (CIT, 0.1 M in water) at a ratio of
2 :1 (v/v). The m/z values are those of the unresolved isotope
envelope of the pseudomolecular ions ([M� H]� ). Quantification of
oligonucleotides was performed with a NanoDrop 1000 spectral
photometer from NanoDrop Technologies (Wilmington, United
States). The UV-VIS spectra were measured on a Perkin Elmer
Lambda 25 spectrometer.

2-Bromo-3-iodo-5-methyl-6-pyridone (10). 2-Bromo-3-iodo-5-meth-
yl-6-pivaloyloxypyridine 9 (500 mg, 1.26 mmol, 1 eq) was dissolved
in acetonitrile (2 mL) and aqueous ammonia (25%, 2 mL) was
added. The reaction mixture was stirred for 3 h at room temper-
ature. After TLC showed full conversion, water (10 mL) was added,
ammonia was removed with a stream of nitrogen, and the
suspension was lyophilized. Pyridone 10 (391 mg, 1.25 mmol, 99%)
was obtained as a colorless solid. TLC (petroleum ether/ethyl
acetate, 5 : 1, v/v): Rf=0.63;

1H-NMR (700 MHz, DMSO-d6): δ (ppm)=
11.81 (br s, 1H), 7.90 (s, 1H), 2.02-2.00 (m, 3H); 13C-NMR (175 MHz,
DMSO-d6): δ (ppm)=161.5, 149.6, 140.0, 120.9, 85.6, 14.5; HRESIMS
(ESI-TOF): m/z calc. for: C6H5BrINO [M+H]+ 313.867, found 313.867.

2-Bromo-3-iodo-5-methyl-6-(pivaloyloxymethoxy)-pyridine (11). Pyr-
idone 10 (103 mg, 0.33 mmol, 1.0 eq) was dissolved in dry DMF
(2 mL) and K2CO3 (0.14 g, 0.98 mmol, 3.0 eq) and chloromethyl
pivalate (76.0 μL, 0.52 mmol, 1.6 eq) were added. After stirring at
room temperature for 16 h, the reaction mixture was filtered
through celite, and it was then washed with ethyl acetate (15 mL).
The combined filtrates were washed with NaHCO3 (sat. aq. solution,
10 mL). The aqueous phase was extracted with ethyl acetate
(10 mL). The combined organic layers were washed with water
(15 mL). After drying over Na2SO4, the solvent was removed in
vacuo. The crude was purified by chromatography on silica (10 g)
with a gradient of ethyl acetate (5–20%) in petroleum ether. Title
compound 11 was obtained as a colorless oil (115 mg, 0.27 mmol,
82%). TLC (petroleum ether/ethyl acetate, 5 :1, v/v): Rf=0.74;

1H-
NMR (400 MHz, CDCl3): δ (ppm)=7.79-7.75 (m, 1H), 6.02 (s, 2H),
2.11-2.09 (m, 3H), 1.20 (s, 9H); 13C-NMR (100 MHz, CDCl3): δ (ppm)=
177.5, 159.1, 150.4, 141.1, 122.1, 89.4, 82.7, 39.0, 27.0, 14.8; HRESIMS:
m/z calc. for: C12H15BrINO3 [M+H]+ 427.935, found 427.935.
Reactions on larger scales gave similar yields.

2-Bromo-5-methyl-6-(pivaloyloxymethoxy)-3-[3’-O-(tert-butyldimeth-
ylsilyl)-2’-deoxy-2’,3’-didehydro-β-D-ribofuranos-1’-yl]-pyridine (16).
Samples of Pd(OAc)2 (43.0 mg, 0.19 mmol, 0.2 eq) and P(PhF5)
(203 mg, 0.38 mmol, 0.4 eq) were dissolved in dry acetonitrile
(4 mL), and the brown solution was stirred for 30 min at room
temperature. Subsequently, glycal 12 (217 mg, 0.94 mmol, 1 eq)
and aglycone 11 (443 mg, 1.04 mmol, 1.1 eq), dissolved in dry
acetonitrile (4 mL), were added, followed by addition of Ag2CO3
(260 mg, 0.94 mmol, 1 eq). The reaction mixture was stirred for 16 h
at room temperature. After TLC showed full conversion, the grey
suspension was filtered through celite and eluted with acetonitrile
(15 mL). The solvent was removed in vacuo. The resulting crude 16
(907 mg) was obtained as a dark brown oil and was used in the
subsequent step without purification. TLC (petroleum ether/diethyl
ether, 1 : 1, v/v): Rf=0.66.

Figure 2. Synthesis of oligonucleotides containing W. A) Sequences prepared
using 3. B) HPLC trace of the crude 15 (C18 column, TEAA buffer/CH3CN,
55 °C) (top), and MALDI-TOF mass spectrum of 15 after HPLC purification in
linear negative mode (bottom).

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2-Bromo-5-methyl-6-(pivaloyloxymethoxy)-3-(3’-dehydro-2’,3’-di-
deoxy-3’-oxo-β-D-ribofuranos-1’-yl)-pyridine (17). In a polypropy-
lene tube, crude 16 (907 mg, 0.94 mmol), obtained as described in
the preceding protocol, was dissolved in dry THF (18 mL). After
addition of 3HF·NEt3 (0.30 mL, 1.89 mmol, 2.0 eq) the reaction
mixture was shaken for 30 min at room temperature. To quench
remaining HF, methoxytrimethylsilane (2 mL) was added, and the
mixture was shaken for 30 min at room temperature. The dark
solution was filtered through celite and eluted with ethyl acetate
(20 mL). After removing the solvent in vacuo, crude 17 (851 mg)
was obtained, which was used without further purification. TLC
(petroleum ether/diethyl ether, 1 : 1, v/v): Rf=0.51.

2-Bromo-5-methyl-6-(pivaloyloxymethoxy)-3-(2’-deoxy-β-D-ribofura-
nos-1’-yl)-6-(triisopropylsilylethynyl)-pyridine (13). Crude 17
(851 mg, 0.94 mmol) was dissolved in a mixture of dry acetonitrile
and acetic acid (3 : 1, v/v,15 mL) and cooled to 0 °C. Then,
NaBH(OAc)3 (300 mg, 1.42 mmol, 1.5 eq) was added, and the
reaction mixture was stirred for 2 h at 0 °C. After TLC showed full
conversion, methanol (5 mL) was added, and the solvent was
removed in vacuo. The crude product was purified by chromatog-
raphy on silica (50 g) with a gradient of methanol (1-5%) in
dichloromethane, yielding 254 mg (0.60 mmol, 64% over 3 steps)
of the desired C-nucleoside 13 as an orange oil. TLC (dichloro-
methane/methanol, 9 : 1, v/v): Rf=0.49;

1H-NMR (400 MHz, CDCl3): δ
(ppm)=7.53 (s, 1H), 6.06-6.00 (m, 2H), 5.32-5.26 (m, 1H), 4.41-4.35
(m, 1H), 4.02-3.95 (m, 1H), 3.85-3.75 (m, 2H), 2.51-2.42 (m, 1H),
2.12(s, 3H), 1.86-1.75 (m, 1H), 1.20-1.16 (m, 9H); 13C-NMR (125 MHz,
CDCl3): δ (ppm)=177.6, 158.6, 138.6, 133.9, 131.6, 120.5, 87.1, 82.8,
78.0, 73.7, 63.5, 42.9, 39.0, 27.04, 26.98, 15.2; HRESIMS: m/z calc. for:
C17H24NO6Br [M+Na]+ 440.068, found 440.069.

3-Methyl-2-(pivaloyloxymethoxy)-5-(2’-deoxy-β-D-ribofuranos-1’-yl)-
6-(triisopropylsilylethynyl)-pyridine (6). A sample of C-nucleoside 13
(139 mg, 0.33 mmol, 1 eq) was dissolved in dry DMF (5 mL) and
transferred to a pressure-stable reaction vessel. Then, Pd(PPh3)2Cl2
(23.0 mg, 0.02 mmol, 0.1 eq), CuI (13.0 mg, 0.07 mmol, 0.2 eq), NEt3
(92.0 μL„ 0.66 mmol, 2 eq) and triisopropylsilylacetylene (0.37 mL,
1.66 mmol, 5 eq) were added, the reaction vessel was provided
with dry DMF (3 mL) and sealed. After stirring for 16 h at 80 °C, the
reaction mixture was filtered through celite and eluted with ethyl
acetate (20 mL). The solvent was removed in vacuo and the crude
product was purified by chromatography on silica (15 g) with a
gradient of methanol (1-5%) in dichloromethane, yielding 105 mg
(0.20 mmol, 61%) of title compound 6 as a light brown oil. TLC
(dichloromethane/methanol, 9 : 1, v/v): Rf=0.60;

1H-NMR (400 MHz,
CDCl3): δ (ppm)=7.53 (s, 1H), 6.10 (d, J=5.3 Hz, 1H), 6.05 (d, J=

5.4 Hz, 1H), 5.56 (dd, J=9.6 Hz, J=6.3 Hz, 1H), 4.39-4.35 (m, 1H),
3.96-3.93 (m, 1H), 3.84-3.75 (m, 2H), 2.40-2.36 (m, 1H), 2.18 (s, 3H),
1.87-1.82 (m, 1H), 1.20-1.06 (m, 30H); 13C-NMR (100 MHz, CDCl3): δ
(ppm)=177.7, 158.9, 136.4, 134.73, 134.69, 122.1, 103.5,95.2, 87.1,
83.0, 76.8, 73.5, 63.4, 43.2, 38.9, 27.7, 27.0, 18.809, 18.802, 15.9, 11.4;
HRESIMS: m/z calc. for: C28H45NO6Si [M+H]+ 520.309, found
520.309.

3-Methyl-5-(2’-deoxy-β-D-ribofuranos-1’-yl)-6-(triisopropylsilyl-
ethynyl)-2-pyridone (5). The C-nucleoside starting material 4
(504 mg, 1.02 mmol, 1 eq) was dissolved in acetonitrile (5 mL), and
aqueous ammonia (25%, 5 mL) was added. The reaction mixture
was stirred for 2 h at room temperature. After TLC showed full
conversion, water (10 mL) was added, remaining ammonia was
removed with a stream of nitrogen, and the suspension was
lyophilized to dryness. Title compound 5 was obtained as a
colorless solid in a yield of 360 mg (0.89 mmol, 87%). TLC (dichloro-
methane /methanol, 9 :1, v/v): Rf=0.34;

1H-NMR (400 MHz, CD3OD):
δ (ppm)=7.69–7.64 (m, 1H), 5.42 (dd, J=10.3 Hz, J=5.5 Hz, 1H),
4.37-4.32 (m, 1H), 3.91–3.86 (m, 1H), 3.75-3.65 (m, 2H), 2.16-2.09 (m,
4H), 1.97-1.84 (m, 1H), 1.22–1.10 (m, 21 H); 13C-NMR (100 MHz,

CD3OD): δ (ppm)=164.9, 138.4, 132.6, 126.6, 124.6, 102.7, 97.7, 89.0,
77.4, 74.1, 63.7, 43.5, 19.1, 16.8, 12.4; HRESIMS: m/z calc. for:
C22H35NO4Si [M+Na]+ 428.223, found 428.223.

3-Methyl-2-(pivaloyloxymethoxy)-5-(2’-deoxy-β-D-ribofuranos-1’-yl)-
6-(triisopropylsilylethynyl)-pyridine (6). Nucleosidic pyridone 5
(360 mg, 0.89 mmol, 1.0 eq) was dissolved in dry DMF (10 mL), and
K2CO3 (184 mg, 1.33 mmol, 1.5 eq) as well as chloromethyl pivalate
(192 μL, 1.33 mmol, 1.5 eq) were added. After stirring at room
temperature for 16 h, the reaction mixture was filtered through
celite and was washed with ethyl acetate (10 mL). The combined
filtrates were washed with water (10 mL). The aqueous phase was
back-extracted with ethyl acetate (3 x 10 mL), and the combined
organic layers were washed with water (3×30 mL). After drying
over Na2SO4, the solvent was removed in vacuo. The crude was
purified by chromatography on silica (30 g) with a gradient of
methanol (1-5%) in dichloromethane. Besides 205 mg (0.39 mmol,
44%) O-alkylated C-nucleoside 6 as a colorless oil, 63.0 mg
(0.12 mmol, 14%) N-alkylated C-nucleoside 7 was obtained as a
colorless solid. Analytical data for 6: TLC (dichloromethane/meth-
anol, 9 :1, v/v): Rf=0.60;

1H-NMR (400 MHz, CDCl3): δ (ppm)=7.53 (s,
1H), 6.10 (d, J=5.3 Hz, 1H), 6.05 (d, J=5.4 Hz, 1H), 5.56 (dd, J=

9.6 Hz, J=6.3 Hz, 1H), 4.39-4.35 (m, 1H), 3.96-3.93 (m, 1H), 3.84-3.75
(m, 2H), 2.40-2.36 (m, 1H), 2.18 (s, 3H), 1.87-1.82 (m, 1H), 1.20-1.06
(m, 30H); 13C-NMR (100 MHz, CDCl3): δ (ppm)=177.7, 158.9, 136.4,
134.73, 134.69, 122.1, 103.5,95.2, 87.1, 83.0, 76.8, 73.5, 63.4, 43.2,
38.9, 27.7, 27.0, 18.809, 18.802, 15.9, 11.4; HRESIMS: m/z calc. for:
C28H45NO6Si [M+H]+ 520.309, found 520.309. Analytic data for 3-
methyl-N-(pivaloyloxymethyl)-5-(2’-deoxy-β-D-ribofuranos-1’-yl)-6-
(triisopropylsilylethynyl)-2-pyridone (7): TLC (dichloromethane/
methanol, 9 : 1, v/v): Rf=0.46;

1H-NMR (700 MHz, CDCl3): δ (ppm)=
7.34 (s, 1H), 6.28–6.04 (m, 2H), 5.46 (dd, J=10.1 Hz, J=5.8 Hz, 1H),
4.46-4.42 (m, 1H), 3.96–3.93 (m, 1H), 3.86–3.77 (m, 2H), 2.21–2.12
(m, 4H), 1.97–1.91 (m, 1H), 1.19–1.09 (m, 30H); 13C-NMR (175 MHz,
CDCl3): δ (ppm)=177.6, 162.3, 135.0, 132.4, 126.0, 124.7, 106.2, 95.4,
87.2, 76.8, 73.6, 70.4, 63.4, 42.5, 39.0, 27.2, 18.779, 18.773, 17.7, 11.3;
HRESIMS: m/z calc. for: C28H45NO6Si [M+Na]+ 542.291, found
542.291.

3-Methyl-2-(pivaloyloxymethoxy)-5-(2’-deoxy-5’-O-(dimethoxytrityl)-
β-D-ribofuranos-1’-yl)-6-(triisopropylsilylethynyl)-pyridine (8). Pro-
tected C-nucleoside 6 (194 mg, 0.37 mmol, 1.0 eq) was coevapo-
rated with dry pyridine (2×5 mL) and dissolved in dry pyridine
(5 mL). Dimethoxytrityl chloride (91.0 mg, 0.27 mmol, 1.1 eq),
previously coevaporated with dry pyridine (3 mL), was dissolved in
dry pyridine (2 mL) and was added. After stirring for 16 h at room
temperature, TLC showed full conversion. The solvent was removed
in vacuo and the crude product was purified by chromatography
on silica [20 g, deactivated with NEt3 (0.5% in dichloromethane)
prior to use] with a gradient of methanol (0-1%) in dichloro-
methane. Title compound 8 was obtained as a colorless glass
(206 mg, 0.25 mmol, 68%). TLC (dichloromethane/methanol, 40 :1,
v/v): Rf=0.75;

1H-NMR (500 MHz, CD3CN): δ (ppm)=7.76 (s, 1H),
7.51-7.18 (m, 9H), 6.89–6.82 (m, 4H), 6.05–6.01 (m, 2H), 5.46 (dd, J=

9.7 Hz, J=5.8 Hz, 1H), 4.36–4.27 (m, 1H), 3.98–3.93 (m, 1H), 3.74 (s,
6H), 3.28–3.20 (m, 2H), 3.17(d, J=3.8 Hz, 1H), 2.33-2.27 (m, 1H), 2.02
(s, 3H), 1.93-1.84 (m, 1H), 1.17–1.13 (m, 30H); 13C-NMR (125 MHz,
CD3CN): δ (ppm)=178.0, 159.7, 146.3, 138.3, 137.3, 137.11, 137.07,
134.9, 131.0, 129.0, 128.8, 127.8, 123.3, 114.0, 104.6, 95.8, 87.4, 87.3,
87.0, 83.3, 77.4, 74.3, 74.2, 65.4, 55.9, 44.0, 39.4, 27.2, 19.1, 12.1;
HRESIMS: m/z calc. for: C49H63NO8Si [M+H]+ 822.440, found
822.440.

3-Methyl-2-(pivaloyloxymethoxy)-5-[2’-deoxy-3’-O-(2-cyanoethyl-
N,N-diisopropylamino)-phosphino-5’-O-(dimethoxytrityl)-β-D-ribo-
furanos-1’-yl]-6-(triisopropylsilylethynyl)-pyridine (3). The C-nucleo-
side 8 (85.0 mg, 103 μmol, 1.0 eq) was coevaporated with
acetonitrile (3×2 mL) and briefly vacuum-dried dried together with

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diisopropylammonium tetrazolide (12.5 mg, 0.07 mmol, 0.7 eq).
Subsequently, dry acetonitrile (1 mL) and 2-cyanoethyl-N,N,N’,N’-
tetraisopropyl phosphorodiamidite (43.0 μL, 0.07 mmol, 1.3 eq)
were added, and the mixture was stirred for 4 h at room temper-
ature. After TLC showed full conversion, the reaction mixture was
chromatographed on silica (7 g), eluting with a mixture of methyl
tert-butyl ether and petroleum ether (3 :1, v/v, with 1% NEt3).
Phosphoramidite 3 was obtained as a colorless glass (98.6 mg,
96.4 μmol, 93%). TLC (petroleum ether/ethyl acetate, 5 : 1, v/v, with
1% NEt3): Rf=0.70;

31P-NMR (121.5 MHz, CD3CN): δ (ppm)=147.73,
146.96; HRESIMS: m/z calc. for: C58H80N3O9PSi [M+H]+ 1022.547,
found 1022.548.

DNA synthesis and deprotection. The desired oligodeoxynucleotide
sequences were synthesized on LCCA controlled pore glass (cpg),
up to the position where the W residue was to be incorporated, by
automated DNA synthesis on a H-2 synthesizer (K&A Laborgeräte,
Schaafheim, Germany) on a 1 μmol scale, using the protocol
recommended by the manufacturer and commercial phosphorami-
dites. The cpg (40 mg, 1 μmol oligonucleotide loading) was dried in
a pear-shaped flask in vacuo. Then, dry phosphoramidite 3
(28.5 mg, 28 μmol) was dissolved in “activator solution” for
automated DNA synthesis (350 μL, 0.25 M 5-(ethylthio)tetrazole in
dry acetonitrile). The solution was immediately added to the cpg,
and the mixture was shaken in a horizontal shaker for 1 h at room
temperature. The supernatant was removed, and the solid support
was washed with dry acetonitrile (800 μL). Subsequently, “cap-A”
(200 μL, 9% acetic anhydride in THF) and “cap-B” (200 μL THF/
pyridine/1-methylimidazole, 80 :10 :10) solutions were added, and
the mixture was shaken for 5 min. The supernatant was removed
and the cpg was washed with dry acetonitrile (800 μL). Then,
“oxidizer solution” (750 μL, 3% iodine in THF /water/pyridine) was
added and the slurry was shaken for 10 min. The supernatant was
again removed, and the solid support was washed with dry
acetonitrile (5×800 μL), followed by drying in vacuo, and back-
transfer to a column for DNA synthesis. The remaining synthesis
cycles were performed by automated DNA synthesis as described
for the first phase of chain assembly, above. Oligonucleotide 14
was thus synthesized with its 5’-terminal DMT group in place. The
cpg was placed in a polypropylene tube and aqueous ammonia
(25%, 1 mL) was added. After 2 h at room temperature, the
supernatant was aspirated, and the cpg was washed with water (4×
300 μL). The ammonia in the combined solutions was removed
with a gentle stream of nitrogen, and the sample was lyophilized.
The resulting crude was purified by HPLC. The DMT group of the
support bound form of oligonucleotide 15 was removed at the end
of the chain assembly. The cpg was then placed in a polypropylene
tube, and aqueous ammonia (25%, 1 mL) was added. After
incubating for 16 h at 55 °C, the supernatant was collected, and the
cpg was washed with water (4 x 300 μL). The ammonia of the
combined aqueous solutions was removed with a stream of
nitrogen directed onto the surface of the solution, and the sample
was lyophilized. Then, TBAF (1 M in THF, 500 μL) was added and
subsequently water (20 μL) was added, in order to fully dissolve the
oligonucleotide. The mixture was shaken for 5 h at room temper-
ature on a horizontal shaker. Then, triethylammonium acetate
buffer (1 M in water, 500 μL) and water (500 μL) were added, and
the THF was removed with a stream of nitrogen. The sample was
lyophilized and purified by HPLC, as described below.

HPLC purification. Reversed-phase HPLC was performed on a
DIONEX ULTIMATE 300 HPLC system (Thermo Fischer Scientific,
Waltham, USA) using an Nucleosil C18 column (250×4.6 mm,
Macherey-Nagel, Düren, Germany). The lyophilized oligonucleotides
were dissolved in water (1 mL) and the solution was filtered (pore
size: 0.45 μm) prior to injection. A gradient of acetonitrile in

triethylammonium acetate buffer (0.1 M, pH 7) was used, at 55 °C
and a flow of 0.6 mL/min.

5’-DMT-TTTTTTTTWTIPSTTT-3’ (14) HPLC: gradient of acetonitrile (1-
50% in 40 min) in triethylammonium acetate buffer (0.1 M, pH 7),
tr=35.7 min. yield: 324 nmol, 32%; MALDI-TOF-MS: m/z: calc. for
C153H196N22O83P11Si [M� H]

� 4054, found 4053.

5’-TTACAAACATWGGCCGCAAA-3’ (15) HPLC: gradient of acetonitrile
(1-35% in 40 min) in triethylammonium acetate buffer (0.1 M,
pH 7), tr= 26.1 min. yield: 479 nmol, 48%; MALDI-TOF-MS: m/z: calc.
for C198H245N77O113P19 [M� H]

� 6100, found 6102.

Acknowledgements

The authors thank Dr. Curtis H. Lam and Stephen H. Strickland for
sharing experimental results, Jianyang Han, Tim Gniech, Dustin
Dittrich and Dr. Eric Kervio for discussions and Deutsche
Forschungsgemeinschaft (DFG) grant RI 1063/18-1 to C.R. for
financial support. Open Access funding enabled and organized by
Projekt DEAL.

Conflict of Interest

The authors declare no conflict of interest.

Data Availability Statement

The data that support the findings of this study are available in
the supplementary material of this article.

Keywords: Base pairing · C-nucleosides · DNA ·
Oligonucleotides · Thymidine analogs

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Manuscript received: May 26, 2022
Revised manuscript received: June 27, 2022
Accepted manuscript online: June 28, 2022

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