Design And Synthesis Of An Azabicyclic Nucleoside Phosphoramidite For Oligonucleotide Antisense Constructs

Article abstract of DOI:10.1080/15257770.2019.1646916

We report the synthesis and biophysical evaluation of an azabicycle dinucleotide with restricted γ, β, and ε torsion angles, featuring the introduction of a piperidine ring that locks the conformation of the nucleoside into an RNA-type nucleic acid. The c

Full text of DOI:10.1080/15257770.2019.1646916

Nucleosides, Nucleotides and Nucleic Acids
ISSN: 1525-7770 (Print) 1532-2335 (Online) Journal homepage: https://www.tandfonline.com/loi/lncn20
Design And Synthesis Of An Azabicyclic Nucleoside
Phosphoramidite For Oligonucleotide Antisense
Constructs
Juan C. Salinas, Punit P. Seth & Stephen Hanessian
To cite this article: Juan C. Salinas, Punit P. Seth & Stephen Hanessian (2019): Design
And Synthesis Of An Azabicyclic Nucleoside Phosphoramidite For Oligonucleotide Antisense
Constructs, Nucleosides, Nucleotides and Nucleic Acids, DOI: 10.1080/15257770.2019.1646916
To link to this article: https://doi.org/10.1080/15257770.2019.1646916
Published online: 05 Aug 2019.
Submit your article to this journal
View Crossmark data
Full Terms & Conditions of access and use can be found at
https://www.tandfonline.com/action/journalInformation?journalCode=lncn20

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
https://doi.org/10.1080/15257770.2019.1646916
Design And Synthesis Of An Azabicyclic Nucleoside
Phosphoramidite For Oligonucleotide Antisense
Constructs
Juan C. Salinasa, Punit P. Sethb , and Stephen Hanessiana
a
ꢀ
ꢀ
ꢀ
Department of Chemistry, Universite de Montreal, Downtown Station, Montreal, P.O. Box 6128,
b
Canada QC H3C 3J7; Department of Medicinal Chemistry, Ionis Pharmaceuticals, 2855 Gazelle
Court, Carlsbad, CA 92010, USA
ABSTRACT
ARTICLE HISTORY
Received 17 March 2019
Accepted 17 July 2019
We report the synthesis and biophysical evaluation of an
azabicycle dinucleotide with restricted c, b, and e torsion
angles, featuring the introduction of a piperidine ring that
locks the conformation of the nucleoside into an RNA-type
nucleic acid. The conceptual basis of the design is predicated
upon the notion that the conformation of the phosphate
group linking two RNA nucleotides can be approximated with
an azabicyclic phosphoramidite which may also benefit from
a unique stereoelectronic effect.
KEYWORDS
antisense; conformational
restriction; azabicyclic
nucleosides
GRAPHICAL ABSTRACT
Introduction
Oligonucleotides modified with conformationally restricted nucleic acid
analogs, represents an important area of research in oligonucleotide thera-
peutics.[1–3] The introduction of one or more modified nucleosides into an
oligonucleotide construct, reshapes the general conformation of the oligo,
which in turn, impacts the affinity for complementary RNA or DNA
strands. The conformationally constrained nucleosides, constitutes some of
the most studied series of modifications. By restricting the pseudorotation
CONTACT Stephen Hanessian
stephen.hanessian@umontreal.ca; Punit P. Seth
pseth@ionisph.com
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lncn.
ß 2019 Taylor & Francis Group, LLC

2
J. C. SALINAS ET AL.
Figure 1. Conceptual design of an azabicyclic nucleoside phosphoramidite A as conformational
surrogate of an RNA dinucleotide.
of the furanose ring, oligonucleotides containing constrained nucleosides
have shown unprecedented affinity as demonstrated by LNA[4–6], TriNA[7],
a-L-TriNA[8]. Over the years, several groups have focused their efforts
in restricting a set of torsion angles in modified nucleosides. Escudier[9]
demonstrated that restriction around a and b torsion angles improved
affinity to complementary RNA and DNA without imposing a C3’-endo
conformation in the furanose ring. In a series of reports, we used a double-
constrain strategy to restrict torsion angles c and d. Once introduced into
oligos, the resulting a-L-TriNA 1 and 2 nucleosides presented enhanced
affinity versus RNA and DNA complementary strands.
In a recent report[10], we synthesized a oxabicyclic nucleic acid phosphonate
as an internucleotide phosphate surrogate analog with restricted c, b, and e
torsion angles. The thermal affinity measurements suggested that the applied
constrain can mimic conformational preferences of RNA-like nucleic acids.
Following a similar strategy, we report here the synthesis and biophysical
evaluation of an azabicycle dinucleotide A (Figure 1) with restricted c, b, and e
torsion angles, featuring the introduction of a piperidine ring that locks the
conformation of the nucleoside into an RNA-type nucleic acid. The conceptual
basis of the design is predicated upon the notion that the conformation of
the phosphate group linking two RNA nucleotides can be approximated
with an azabicyclic phosphoramidite which may also benefit from a unique
stereoelectronic effect as depicted in Figure 1.
The synthesis of a 20-deoxypiperidine related nucleoside has been
previously reported by Wang[11,12] by using commercially available 30-azido-

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
3
3’-deoxythymidine (AZT). In our target nucleoside A, the furanose ring is
functionalized at the 2’-position with a 2’-OMe group which is intended to
enforce the furanose ring into a C3’-endo conformation. In order to facilitate
the installation of the different functionalities in A, we developed a carbohy-
drate-based route that secured stereocontrol in key transformations.
We started with known diol 2 which is readily available from diacetone-
D-glucose (Scheme 1).[13,14] Selective tosylation of the primary alcohol in 2
and subsequent base-mediated cyclization provided epoxide 3 in 59% yield.
Opening of epoxide 3 with lithiated 1,3-dithiane[15,16] produced a second-
ary alcohol which was protected with 2-naphthylmethyl bromide to give ether
4. Unmasking of the dithiane in 4 with MeI/K2CO3, led to an aldehyde that
was reduced together with the azide to give aminoalcohol 5. In order to avoid
several steps involving activation/protection/deprotection for the formation
of piperidine 6, we explore the possibility to use Mitsunobu conditions, being
cognizant that there are only few precedents in literature for the formation of
phosphine-mediated azacycles.[17,18] Gratifyingly, piperidine formation under
standard Mitsunobu conditions with subsequent protection with DIPEA/
BnBr[19] gave azabicyclic sugar 6 in good yield. The thymine nucleobase was
installed following a three steps sequence starting from TFA[20] mediated
cleavage of the 1,2-isopropylidene group, acetylation of the anomeric mixture
of alcohols (not shown) and Vorbru€ggen glycosylation[21,22] to give nucleo-
side 7. Protection of the nucleobase under phase-transfer conditions resulted
in the installation of a PMB with concomitant cleavage of the acetate group
and subsequent methylation at the 2’-position providing nucleoside 8.
Removal of the N-benzyl group in 8 was problematic under several
hydrogenolysis conditions. Fortunately, the exchange of the benzyl for an
Scheme 1. Synthesis of azabicyclic nucleoside 10.

4
J. C. SALINAS ET AL.
Scheme 2. Synthesis of azabicyclic phosphoramidite 15.
alloc group was successfully achieved by refluxing nucleoside 8 with allyl
chloroformate in toluene. Pd-mediated[23] cleavage of the alloc carbamate
released the free amine which was later protected as a trifluoroacetamide[24]
providing nucleoside 9. At this juncture, we cleaved the 50-O naphthyl
ether with DDQ[25] and the N-PMB using CAN.[26] Esterification of the
50-secondary alcohol afforded the levulinic ester[27] at the 50-position
(Scheme 2). Selective cleavage of the N-trifluoroacetyl group in 10 provided
the free amine 11, which was engaged in an Atherton-Todd[12] reaction
with H-phosphonate 14 prepared in two steps from the known[28] nucleoside
13. The resulting nucleotide was deprotected using TBAF giving phosphorami-
date 12. Conversion of the dimer 12 to the corresponding phosphoramidite
15 was accomplished using 2-cyanoethyl tetraisopropylphosphordiamidite.
Unfortunately, the dimer phosphoramidite 15 underwent spontaneous air
oxidation after column chromatography to yield the phosphoramidate dimer
16, which was not suitable for incorporation into oligonucleotides.
In conclusion, we describe the design and synthesis of an azabicycle
dinucleotide with restricted c, b, and e torsion angles, featuring the introduction

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
5
of a piperidine ring that locks the conformation of the nucleoside into an
RNA-type nucleic acid. Further incorporation into oligonucleotides will
reveal its utility for oligonucleotide medicinal chemistry.
Experimental section
General Information: All non-aqueous reactions were run in oven dried
(120ꢀC) glassware under a positive pressure of argon, with exclusion of
moisture from reagents and glassware, using standard techniques for
manipulating air-sensitive compounds, unless otherwise stated. Anhydrous
tetrahydrofuran, diethyl ether, toluene, and CH2Cl2 were obtained by pass-
ing these solvents through activated columns of alumina, while all other
solvents were used as received from chemical suppliers. Reagents were
purchased and used without further purification. Yields refer to chromato-
graphically and spectroscopically (1H NMR) homogeneous material, unless
otherwise stated. Reactions were monitored by thin-layer chromatography
(TLC) carried out on 0.25 mm Machery-Nagel silica plates (SIL 60, G-25,
UV254) that were visualized using a UVP UVG-11 compact UV lamp
(254 nm) and developed with an aqueous solution of cerium ammonium
molybdate, or an ethanolic solution of p-anisaldehyde. Flash chromatog-
raphy was performed using SiliaFlash P60 40-63 lm (230-400 mesh) silica
gel. NMR spectra were recorded on Bruker AV-300, ARX-400, or AV-400
instruments, calibrated using residual undeuterated solvent as an internal
reference (CHCl3, d ¼ 7.26 ppm), and reported in parts per million relative to
trimethylsilane (TMS d ¼ 0.00 ppm) as follows: chemical shift (multiplicity,
coupling constant (Hz), integration). The following abbreviations were used
to explain multiplicities: s ¼ singlet, d ¼ doublet, t ¼ triplet, q ¼ quartet,
m¼ multiplet, br ¼ broad, dd ¼ doublet of doublets, dt ¼ doublet of triplets,
ddt ¼ doublet of doublet of triplets. High resolution mass spectra (HRMS)
ꢀ
ꢀ
were recorded at the Center Regional de Spectrometrie de Masse de
ꢀ
ꢀ
l’Universite de Montreal on an Agilent LC-MSD TOF mass spectrometer by
electrospray ionization time of flight reflectron experiments. IR spectra were
recorded on a Perkin Elmer Spectrum One spectrometer and are reported in
reciprocal centimeters (cmꢁ1).

6
J. C. SALINAS ET AL.
Diol 2
Pyridine (38 mL, 0.473 mol) was added to an stirred solution of 1,2:5,6-di-
O-isopropylidene-a-D-glucofuranose 1 (50.0 g, 0.192 mol) in CH2Cl2
(800 mL) at 0ꢀC. Trifluoromethanesulfonic anhydride (33 mL, 0.196 mol)
was added to the previous solution during a period of 30 min keeping the
temperature under 5ꢀC. The coldbath was removed and the reaction mix-
ture was stirred for 3 h. The organic phase was washed with 1 M HCl
(2 ꢂ 100 mL) then dried over Na2SO4. The solvent was removed under
reduced pressure and coevaporated with toluene (2 ꢂ 50 mL) to furnish the
corresponding triflate. Crude product was dissolved in anhydrous DMF
(250 mL), then mixed subsequently with tetrabutyl ammonium chloride
(270 mg, 0.97 mmol) and sodium azide (25 g, 0.38 mol). The reaction mix-
ture was heated at 60ꢀC for 4 h and then cooled down. The organic phase
was partitioned with the addition of water (750 mL) and extracted with
diethyl ether (4 ꢂ 100 mL). The combined organic extractions were dried
over Na2SO4, filtered and concentrated under reduced pressure. A mixture
of methanol (210 mL), water (260 mL), acetic acid (160 mL) was added to
the crude azide and warmed to 60ꢀC for 6 h, then cooled down. The mix-
ture was neutralized via addition of solid sodium bicarbonate followed by
extraction with ethyl acetate (3 ꢂ 200 mL). The combined organic extracts
were dried over Na2SO4, filtered and concentrated under reduced pressure.
The residue was purified by flash chromatography (7:3 ethyl acetate/hex-
anes) to give diol 2 as an oil (18.6 g, 40% over 3 steps); 1H NMR
(400 MHz, CDCl3) d 5.75 (d, J ¼ 3.7 Hz, 1 H), 4.72 – 4.67 (m, 1 H), 4.00
(dd, J ¼ 9.3, 4.1 Hz, 1 H), 3.92 (dd, J ¼ 7.2, 3.7 Hz, 1 H), 3.65 (dd, J ¼ 10.3,
5.4 Hz, 2 H), 3.56 (dd, J ¼ 9.3, 4.8 Hz, 1 H), 1.51 (s, 3 H), 1.30 (s, 3 H); 13C
NMR (101 MHz, CDCl3) d 113.22, 104.04, 80.57, 77.84, 71.57, 62.96, 60.26,
26.41, 26.38; HRMS (ESI) calc’d for C9H15N3NaO5 [M þ Na]þ m/
z ¼ 268.09039, found ¼ 268.08939; FT-IR (azide) ¼ 2109.0 cmꢁ1.
Epoxide 3
4-(dimethylamino)pyridine (930 mg, 7.58 mmol), and p-toluensulfonyl
chloride (17.5 g, 90.3 mmol) were added sequentially to an stirred solution
of diol 2 (18.6 g, 75.8 mmol) in pyridine (200 mL). After 6 h the reaction

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
7
was quenched with 1 M HCl (300 mL) and the resulting solution was
extracted with ethyl acetate (4ꢂ 50 mL). The combined organic extracts
were dried over Na2SO4, filtered and concentrated under reduced pressure.
The crude tosylate was dissolved in methanol (300mL) and cooled down to
0ꢀC. A 0.5 M solution of sodium methoxide (150 mmol, 300 mL) was added
to the previous solution and let to warm up to room temperature for 1 h.
The reaction was carefully quenched with water and the methanol was
removed under reduced pressure. The remaining aqueous phase was
extracted with ethyl acetate (4ꢂ 50 mL). The combined organic extracts
were dried over Na2SO4, filtered and concentrated under reduced pressure.
The residue was purified by flash chromatography (15:85 ethyl acetate/hex-
1
anes) to give epoxide 3 as an oil (8.9 g, 52% over 2 steps); H NMR
(400 MHz, CDCl3) d 5.79 (d, J ¼ 3.7 Hz, 1 H), 4.71 (t, J ¼ 4.2 Hz, 1 H), 4.14
(dd, J ¼ 9.5, 3.6 Hz, 1 H), 3.29 (dd, J ¼ 9.5, 4.7 Hz, 1 H), 3.21 (dd, J ¼ 6.7,
3.6 Hz, 1 H), 2.84 (t, J ¼ 4.5 Hz, 1 H), 2.79 – 2.75 (m, 1 H), 1.54 (s, 3 H), 1.33
(s, 3 H); 13C NMR (101 MHz, CDCl3) d 113.41, 104.33, 80.44, 77.61, 60.64,
50.47, 44.74, 26.59, 26.56; HRMS (ESI) calc’d for C9H13N3NaO4 [M þ Na]þ
m/z ¼ 250.07983, found ¼ 250.08086, calc’d for C9H17N4O4 [Mþ NH4]þ m/
z ¼ 245.12443, found ¼ 245.12549; FT-IR (azide) ¼ 2105.5 cmꢁ1.
Alcohol 3a
To a cooled (ꢁ20ꢀC) solution of 1,3-dithiane (6.25 g, 50.9 mmol) in THF
(200 mL), was added dropwise a 1.6 M solution of n-butyllithium (31.8 mL,
50.9 mmol), then stirred at the same temperature for 0.5 h. A previously
prepared solution of epoxide 3 (8.9 g, 39.2 mmol) in THF (200 mL) was
added dropwise keeping the temperature at ꢁ20ꢀC. The reaction mixture
was allowed to warm to 0ꢀC and then DMPU (24.0 mL, 198 mmol) was
added slowly. After stirring for 3 h at room temperature, the reaction
mixture was quenched with NH4Cl saturated and the aqueous layer was
extracted with ethyl acetate (3 ꢂ 100 mL). The combined organic extracts
were dried over Na2SO4, filtered and concentrated under reduced pressure.
The residue was purified by flash chromatography (2:8 ethyl acetate/
1
hexanes) to give alcohol 3a as an oil (6.8 g, 50%); H NMR (400 MHz,
CDCl3) d 5.79 (d, J ¼ 3.6 Hz, 1 H), 4.78 – 4.71 (m, 1 H), 4.27 (dd, J ¼ 9.4,

8
J. C. SALINAS ET AL.
4.9 Hz, 2 H), 4.01 (dd, J ¼ 9.3, 3.4 Hz, 1 H), 3.57 (dd, J ¼ 9.3, 4.8 Hz, 1 H),
2.96 – 2.82 (m, 4 H), 2.43 (s, 1 H), 2.18 – 1.83 (m, 4 H), 1.58 (s, 3 H), 1.36
(s, J ¼ 11.6 Hz, 3 H); 13C NMR (101 MHz, CDCl3) d 113.39, 104.11, 80.98,
80.12, 67.46, 59.50, 43.75, 37.65, 30.17, 29.79, 26.63, 26.61, 25.99; HRMS
(ESI) calc’d for C13H22N3O4S2 [M þ H]þ m/z ¼ 348.10462, found ¼
348.10428; FT-IR (azide) ¼ 2102.0 cmꢁ1.
Azide 4
Sodium hydride (1.17 g, 29.4 mmol) was added to a stirred solution of alco-
hol 3a (6.8 g, 19.6 mmol) in DMF (100 mL) at ̶
20ꢀC. After stirring at the
same temperature for 20 min, tetrabutylamonium iodide (730 mg,
1.96 mmol) and 2-(bromomethyl)-naphthalene (6.62 g, 29.4 mmol) were
added sequentially. The reaction mixture was warmed to 0ꢀC and stirred
for 1 h. The reaction was carefully quenched with water (400 mL) and the
aqueous layer was extracted with diethyl ether (3 ꢂ 100 mL). The combined
organic extracts were dried over Na2SO4, filtered and concentrated under
reduced pressure. The residue was purified by flash chromatography (1:9
1
ethyl acetate/hexanes) to give azide 4 as an oil (7.7 g, 81%); H NMR
(400 MHz, CDCl3) d 7.88 – 7.78 (m, 4 H), 7.53 – 7.44 (m, 3 H), 5.76 (d,
J ¼ 3.7 Hz, 1 H), 4.93 (d, J ¼ 11.8 Hz, 1 H), 4.83 (d, J ¼ 11.8 Hz, 1 H), 4.72 –
4.67 (m, 1 H), 4.28 – 4.14 (m, 2 H), 4.07 (dd, J ¼ 10.2, 4.2 Hz, 1 H), 3.62
(dd, J ¼ 9.3, 4.8 Hz, 1 H), 2.86 – 2.62 (m, 3 H), 2.55 – 2.44 (m, 1 H), 2.23 –
2.11 (m, 1 H), 2.07 – 1.88 (m, 2 H), 1.88 – 1.72 (m, 1 H), 1.59 (s, 3 H), 1.37
(s, 3 H); 13C NMR (101 MHz, CDCl3) d 135.96, 133.24, 132.92, 128.07,
127.91, 127.63, 126.71, 126.13, 126.07, 125.93, 113.06, 103.82, 80.66, 80.42,
74.52, 74.44, 59.63, 43.66, 37.04, 30.18, 29.53, 26.51, 26.47, 25.82; HRMS
(ESI) calc’d for C24H30N3O4S2 [M þ H]þ m/z ¼ 488.16722, found ¼
488.165960; FT-IR (azide) ¼ 2105.0 cmꢁ1

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
9
Aldehyde 4a
Iodomethane (10.0 mL, 157.9 mmol) and calcium carbonate (7.9 g,
78.9 mmol) were added sequentially to a stirred solution of azide 4 (7.7 g,
15.8 mmol) in 10:1 acetonitrile/water (350 mL). The resulting mixture was
warmed to 45ꢀC for 24 h. The reaction mixture was partitioned with the
addition of water (100 mL) and extracted with CH2Cl2 (3 ꢂ 100 mL). The
combined organic extractions were dried over Na2SO4, filtered and con-
centrated under reduced pressure. The residue was purified by flash chro-
matography (2:8 ethyl acetate/hexanes) to give aldehyde 4a as an oil
1
(3.8 g, 61%); H NMR (400 MHz, CDCl3) d 9.76 (s, 1 H), 7.88 – 7.79 (m,
3 H), 7.76 (s, 1 H), 7.54 – 7.39 (m, 3 H), 5.78 (d, J ¼ 3.6 Hz, 1 H), 4.91 –
4.80 (m, 2 H), 4.78 – 4.70 (m, 1 H), 4.39 (dt, J ¼ 7.8, 3.8 Hz, 1 H), 4.19
(dd, J ¼ 9.4, 3.2 Hz, 1 H), 3.58 (dd, J ¼ 9.4, 4.7 Hz, 1 H), 2.89 (ddd,
J ¼ 17.4, 8.3, 1.9 Hz, 1 H), 2.75 – 2.64 (m, 1 H), 1.58 (s, 3 H), 1.37 (s,
3 H); 13C NMR (101 MHz, CDCl3) d 199.76, 135.40, 133.33, 133.17,
128.40, 128.06, 127.83, 126.89, 126.33, 126.17, 126.00, 113.46, 104.00,
80.79, 79.81, 74.09, 73.03, 60.51, 45.53, 26.65, 26.61; HRMS (ESI) calc’d
for C21H23N3O5Na [M þ Na]þ m/z ¼ 420.15299, found ¼ 420.15313; FT-
IR (azide) ¼ 2107.4 cmꢁ1
Aminoalcohol 5
Lithium aluminum hydride (1.45 g, 38.3 mmol) was added portionwise to a
stirred solution of aldehyde 4a (3.8 g, 9.6 mmol) in diethyl ether (160 mL)
at 0ꢀC. After stirring for 20 min at the same temperature, the reaction mix-
ture was carefully quenched with water and subsequently treated with 1 M
NaOH (100 mL). The formed solid was filtrated through a celite pad, the
organig layer was separated and the remaining aqueous layer was extracted
with CH2Cl2 (4 ꢂ 50 mL). The combined organic extractions were dried
over Na2SO4, filtered and concentrated under reduced pressure. The resi-
due was purified by flash chromatography (0% to 6% MeOH in CH2Cl2) to
1
give aminoalcohol 5 as an oil (3.5 g, 98%); H NMR (400 MHz, CDCl3) d
7.87 – 7.75 (m, 8 H), 7.52 – 7.42 (m, 6 H), 5.77 (d, J ¼ 3.7 Hz, 2 H), 4.81 (s,
4 H), 4.50 (dd, J ¼ 4.9, 3.8 Hz, 2 H), 3.95 – 3.87 (m, 4 H), 3.84 – 3.67 (m,
4 H), 3.37 – 3.29 (m, 2 H), 2.23 – 1.86 (m, 11 H), 1.53 (s, 6 H), 1.35 (s,

10
J. C. SALINAS ET AL.
6 H); 13C NMR (101 MHz, CDCl3) d 135.75, 133.38, 133.14, 128.46, 128.05,
127.84, 126.95, 126.33, 126.14, 126.07, 112.23, 104.08, 83.22, 81.32, 78.24,
73.11, 59.51, 55.71, 34.02, 26.95, 26.68; HRMS (ESI) calc’d for C21H28N1O5
[M þ H]þ m/z ¼ 374.1962, found ¼ 374.1967;
Piperidine 5a
To a solution of triphenylphosphine (4.92 g, 18.7 mmol) and aminoalcohol
5 (3.5 g, 9.4 mmol) in CH2Cl2 (500 mL), diisopropyl azodicarboxylate
(3.7 mL, 18.7 mmol) was added slowly at 0ꢀC. The cold bath was removed
and the reaction mixture was stirred for 1 h. The reaction mixture was
pre-adsorbed in silica and purified by flash chromatography (20% acetone
in CH2Cl2) to give piperidine 5a as an oil (2.06 g, 62%); 1H NMR
(400 MHz, CDCl3) d 7.86 – 7.74 (m, 4 H), 7.54 – 7.39 (m, 3 H), 5.84
(d, J ¼ 3.4 Hz, 1 H), 4.91 (d, J ¼ 12.5 Hz, 1 H), 4.77 (d, J ¼ 12.5 Hz, 1 H),
4.60 (t, J ¼ 3.6 Hz, 1 H), 4.27 (s, 1 H), 3.70 (dd, J ¼ 10.1, 2.0 Hz, 1 H), 3.17
(dd, J ¼ 10.1, 4.2 Hz, 1 H), 3.04 (td, J ¼ 13.1, 2.6 Hz, 1 H), 2.91 (dd, J ¼ 13.3,
4.3 Hz, 1 H), 1.89 (d, J ¼ 14.2 Hz, 1 H), 1.54 (s, J ¼ 9.3 Hz, 3 H), 1.50 – 1.41
(m, 1 H), 1.33 (s, 3 H); 13 C NMR (75 MHz, CDCl3) d 136.32, 133.27,
132.91, 128.08, 127.88, 127.69, 126.08, 126.05, 125.79, 125.61, 112.60,
104.62, 78.71, 78.07, 72.22, 57.34, 41.57, 30.50, 26.28, 26.06; HRMS (ESI)
calc’d for C21H26NO4 [M þ H]þ m/z ¼ 356.185635, found ¼ 356.18594.
Amine 6
Benzyl bromide (0.76 mL, 6.4 mmol) and K2CO3 (0.88 g, 6.4 mmol) were
added sequentially to a stirred solution of piperidine 5a (2.06 g, 5.80 mmol)
in ethanol (23 mL) at room temperature. After stirring for 4 h, the solvent

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
11
was removed under reduced pressure, and the residue dissolved in water
(100 mL). The aqueous phase was extracted with CH2Cl2 (3 ꢂ 50 mL). The
combined organic extractions were dried over Na2SO4, filtered and concen-
trated under reduced pressure. The residue was purified by flash chroma-
tography (2:8 Ethyl acetate/hexane) to give amine 6 as an oil (1.45 g, 56%);
1H NMR (400 MHz, CDCl3) d 7.90 – 7.77 (m, 4 H), 7.54 – 7.44 (m, 3 H),
7.42 – 7.27 (m, 5 H), 5.90 (d, J ¼ 3.5 Hz, 1 H), 4.94 (d, J ¼ 12.6 Hz, 1 H),
4.85 – 4.73 (m, 2 H), 4.20 – 4.13 (m, 2 H), 4.08 (dd, J ¼ 9.9, 2.4 Hz, 1 H),
3.39 (d, J ¼ 12.7 Hz, 1 H), 2.76 – 2.63 (m, 2 H), 2.41 – 2.28 (m, 1 H), 1.87
(dd, J ¼ 14.5, 2.5 Hz, 1 H), 1.71 – 1.57 (m, 4 H), 1.40 (s, 3 H); 13C NMR
(101 MHz, CDCl3) d 137.47, 136.52, 133.28, 132.89, 129.78, 128.19, 128.02,
127.89, 127.68, 127.20, 126.01, 125.72, 125.63, 112.49, 104.73, 78.88, 77.54,
71.97, 71.38, 63.86, 59.59, 47.42, 29.01, 26.53, 26.03; HRMS (ESI) calc’d for
C28H31NO4 [M þ H]þ m/z ¼ 446.23258, found ¼ 446,23335.
Nucleoside 7
Amine 6 (1.45 g, 3.32 mmol) was dissolved in a 3:2 mixture of trifluoroace-
tic acid/water (20 mL) and heated to 60ꢀC for 4 h, then the solvent was
removed under reduced pressure and co-evaporated with toluene. The
crude residue was dissolved in pyridine (20 mL) followed by slow addition
of acetic anhydride (3 mL). The resulting solution was stirred for 1 h, then
the solvent was removed under reduced pressure and co-evaporated with
toluene and the crude diacetate was dissolved in anhydrous 1,2-dichloro-
ethane (5 mL). N,O-bis(trimethylsilyl)acetamide (8.2 mL, 33.3 mmol) was
added to an stirred solution of thymine (1.26 g, 9.99 mmol) in 1,2-dichloro-
ethane (17 mL) and the resulting suspension was heated at 80ꢀC. After 1 h,
the reaction mixture was cooled down to 0ꢀC and the previously prepared
solution of the diacetate was transferred via cannula followed by addition
of trimethylsilyl trifluoromethanesulfnate (1.81 mL, 9.99 mmol). The result-
ing solution was heated to 60ꢀC for 2 h, then quenched via addition of
saturated solution of NaHCO3. The aqueous layer was extracted with with
CH2Cl2 (3 ꢂ 50 mL). The combined organic extractions were dried over
Na2SO4, filtered and concentrated under reduced pressure. The residue was

12
J. C. SALINAS ET AL.
purified by flash chromatography (1:1 ethyl acetate/hexane) to give nucleo-
1
side 7 as an oil (0.99 g, 54% over 3 steps); H NMR (400 MHz, CDCl3) d
9.46 (s, 1 H), 7.90 – 7.76 (m, 4 H), 7.62 (d, J ¼ 1.1 Hz, 1 H), 7.53 – 7.43 (m,
3 H), 7.35 – 7.20 (m, 7 H), 5.95 (s, 1 H), 5.59 (d, J ¼ 4.3 Hz, 1 H), 4.86 (d,
J ¼ 11.6 Hz, 1 H), 4.73 (d, J ¼ 11.6 Hz, 1 H), 4.31 (s, 1 H), 4.15 (dd, J ¼ 10.5,
2.5 Hz, 1 H), 4.06 (d, J ¼ 13.0 Hz, 1 H), 3.15 (d, J ¼ 12.9 Hz, 1 H), 2.88 (dd,
J ¼ 10.5, 4.3 Hz, 1 H), 2.76 – 2.68 (m, 1 H), 2.36 – 2.26 (m, 1 H), 2.20 (s,
3 H), 2.07 – 1.97 (m, 1 H), 1.67 – 1.51 (m, 1 H), 1.23 (s, 3 H); 13C NMR
(101 MHz, CDCl3) d 169.28, 164.03, 150.03, 137.81, 135.43, 135.27, 133.29,
133.11, 128.89, 128.53, 128.34, 127.80, 127.75, 127.26, 126.56, 126.42,
126.20, 125.63, 110.63, 89.57, 81.14, 74.26, 72.21, 71.49, 60.76, 60.15, 47.69,
27.32, 20.93, 11.80; HRMS (ESI) calc’d for C32H34N3O6 [M þ H]þ m/
z ¼ 556.244212, found ¼ 556.24490.
Nucleoside 7a
Tetrabutylammonium iodide (49 mg, 0.13 mmol) and p-methoxybenzyl
chloride (0.12 mL, 0.86 mmol) were added to a stirred solution of nucleo-
side 7 (367 mg, 0.66 mmol) in CH2Cl2 (3.3 mL). To the previous solution
was added K2CO3 (460 mg, 3.3 mmol) in water (3.3 mL), and the resulting
biphasic mixture was stirred vigorously for 3 h. The reaction mixture was
diluted with CH2Cl2 (10 mL) and the layers were separated. The aqueous
layer was extracted with CH2Cl2 (3 ꢂ 10 mL). The combined organic
extractions were dried over Na2SO4, filtered and concentrated under
reduced pressure. The residue was purified by flash chromatography (4:6
1
Ethyl acetate/hexane) to give nucleoside 7a as an oil (270 mg, 60%); H
NMR (400 MHz, CDCl3) d 7.89 – 7.77 (m, 4 H), 7.67 (d, J ¼ 1.0 Hz, 1 H),
7.55 – 7.42 (m, 5 H), 7.37 – 7.27 (m, 5 H), 6.85 – 6.77 (m, 2 H), 5.91 (s,
1 H), 5.03 (q, J ¼ 13.5 Hz, 2 H), 4.87 (d, J ¼ 11.7 Hz, 1 H), 4.73 (d,
J ¼ 11.7 Hz, 1 H), 4.34 – 4.26 (m, 2 H), 4.20 (dd, J ¼ 10.4, 2.4 Hz, 1 H),
4.00 (d, J ¼ 12.9 Hz, 1 H), 3.75 (s, 3 H), 3.31 – 3.14 (m, 2 H), 2.83 – 2.72
(m, 2 H), 2.42 – 2.29 (m, 1 H), 2.08 – 1.97 (m, 1 H), 1.66 – 1.51 (m, 1 H),
1.26 (d, J ¼ 0.8 Hz, 3 H); 13C NMR (101 MHz, CDCl3) d 163.46, 159.14,
150.88, 137.46, 135.55, 133.68, 133.35, 133.16, 131.04, 129.31, 129.11,

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
13
128.59, 127.87, 127.81, 127.62, 126.50, 126.46, 126.23, 125.59, 113.74,
109.58, 92.07, 80.73, 74.18, 72.47, 71.39, 61.59, 59.98, 55.30, 47.78, 43.76,
27.34, 12.62; HRMS (ESI) calc’d for C38H40N3O6 [M þ H]þ m/
z ¼ 634.29116, found ¼ 634.29024.
Nucleoside 8
To a stirred solution of 7a (270 mg, 0.426mmol) in THF (2.1 mL) at 0ꢀC
was added 60% sodium hydride (26 mg, 0.85 mmol). After stirring at 0ꢀC for
30min, methyl iodide (54lL, 0.85 mmol) was added and the reaction mix-
ture was warmed to room temperature and stirred overnight. The reaction
mixture was quenched with water (10 mL) and the resulting mixture was
extracted with CH2Cl2 (3ꢂ 10 mL). The combined organic extractions were
dried over Na2SO4, filtered and concentrated under reduced pressure. The
residue was purified by flash chromatography (4:6 Ethyl acetate/hexane) to
1
give nucleoside 8 as a white foam (260 mg, 94%); H NMR (400 MHz,
CDCl3) d 7.88 – 7.73 (m, 5 H), 7.54 – 7.27 (m, 11H), 6.84 (d, J ¼ 8.4 Hz,
2 H), 6.03 (s, 1 H), 5.11 (d, J ¼ 13.6Hz, 1 H), 5.01 (d, J ¼ 13.6 Hz, 1 H), 4.86
(d, J ¼ 11.6 Hz, 1 H), 4.70 (d, J ¼ 11.6 Hz, 1 H), 4.33 – 4.19 (m, 2 H), 3.95 (d,
J ¼ 12.6Hz, 1 H), 3.84 (d, J ¼ 4.1 Hz, 1 H), 3.77 (s, 3 H), 3.64 (s, 3 H), 3.27
(d, J ¼ 12.6 Hz, 1 H), 2.80 – 2.60 (m, 2 H), 2.32 (dd, J ¼ 23.5, 12.4Hz, 1 H),
2.04 (d, J ¼ 16.2 Hz, 1 H), 1.69 – 1.56 (m, 1 H), 1.18 (s, J ¼ 11.7 Hz, 3 H); 13C
NMR (101 MHz, CDCl3) d 163.41, 159.10, 150.70, 137.43, 135.49, 133.77,
133.31, 133.13, 130.77, 129.54, 129.32, 128.55, 128.33, 127.82, 127.76, 127.40,
126.58, 126.41, 126.18, 125.64, 113.73, 109.50, 88.71, 83.22, 80.97, 72.59,
71.18, 60.91, 60.06, 58.13, 55.26, 47.55, 43.62, 27.12, 12.45; HRMS (ESI)
calc’d for C39H42N3O6 [M þ H]þ m/z ¼ 648.30681, found ¼ 648.30851.

14
J. C. SALINAS ET AL.
Nucleoside 8a
Allyl chloroformate (2 mL, 28.3 mmol) was added to a stirred solution of
nucleoside 8 (260 mg, 0.40 mmol) in toluene (10 mL) and the resulting
solution was stirred for 2 days at 100ꢀC. After the reaction was complete,
the solvent was removed under reduced pressure and the crude residue was
passed through a silica pad eluted with ethyl acetate to give nucleoside 8a
1
(184 mg, 72%).; H NMR (400 MHz, CDCl3) d 7.86 – 7.76 (m, 4 H), 7.63
(d, J ¼ 1.1 Hz, 1 H), 7.52 – 7.38 (m, 5 H), 6.85 – 6.78 (m, 2 H), 5.99 – 5.88
(m, 2 H), 5.37 – 5.19 (m, 2 H), 5.07 (d, J ¼ 13.6 Hz, 1 H), 4.97 (d,
J ¼ 13.6 Hz, 1 H), 4.87 (d, J ¼ 11.5 Hz, 1 H), 4.74 (d, J ¼ 11.5 Hz, 1 H), 4.69
– 4.61 (m, 1 H), 4.56 (ddt, J ¼ 13.2, 5.7, 1.3 Hz, 1 H), 4.32 (dd, J ¼ 17.7,
3.1 Hz, 2 H), 4.16 (dd, J ¼ 11.0, 2.6 Hz, 1 H), 3.93 (ddd, J ¼ 13.0, 4.9, 3.0 Hz,
1 H), 3.76 (s, J ¼ 3.6 Hz, 3 H), 3.63 (s, 3 H), 3.36 (dd, J ¼ 11.0, 4.1 Hz, 1 H),
3.26 (td, J ¼ 12.7, 3.5 Hz, 1 H), 2.13 (dq, J ¼ 14.6, 3.2 Hz, 1 H), 1.82 – 1.70
(m, 1 H), 1.16 (d, J ¼ 1.0 Hz, 3 H); 13C NMR (101 MHz, CDCl3) d 163.29,
159.05, 156.51, 150.60, 135.03, 133.28, 133.27, 133.14, 132.56, 130.64,
129.20, 128.62, 127.79, 127.75, 126.62, 126.48, 126.27, 125.52, 118.28,
113.71, 109.52, 88.77, 84.06, 80.11, 77.10, 71.56, 71.54, 66.55, 57.92, 55.23,
53.30, 43.60, 40.89, 26.83, 12.44; HRMS (ESI) calc’d for C36H40N3O8
[M þ H]þ m/z ¼ 642.28099, found ¼ 642.28125.
Nucleoside 8 b
Morpholine (27 lL, 0.31 mmol) and Pd(PPh3)4 (9 mg, 7.8 lmol) were added
sequentially to an stirred solution of nucleoside 8a (98 mg, 0.15 mmol) in
THF (1 mL). After 30 min, the reaction mixture was diluted with CH2Cl2
and silica gel was added to the reaction mixture. The volatiles were
removed under reduced pressure and the dry residue was purified by flash
chromatography (0% to 5% MeOH in CH2Cl2) to give nucleoside 8 b as a
1
white foam (87 mg, 99%); H NMR (400 MHz, CDCl3) d 7.84 – 7.76 (m,
4 H), 7.65 (d, J ¼ 1.0 Hz, 1 H), 7.52 – 7.36 (m, 5 H), 6.80 (d, J ¼ 8.7 Hz,
2 H), 5.93 (s, 1 H), 5.07 (d, J ¼ 13.6 Hz, 1 H), 4.97 (d, J ¼ 13.6 Hz, 1 H), 4.88
(d, J ¼ 11.6 Hz, 1 H), 4.74 (d, J ¼ 11.6 Hz, 1 H), 4.41 (s, 1 H), 3.87 (dd,

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
15
J ¼ 10.8, 2.5 Hz, 1 H), 3.76 (s, 3 H), 3.74 – 3.70 (m, 2 H), 3.65 (d, J ¼ 4.6 Hz,
1 H), 3.61 (s, 3 H), 3.19 (dd, J ¼ 10.8, 4.6 Hz, 1 H), 2.07 (d, J ¼ 13.8 Hz,
1 H), 1.53 – 1.41 (m, 1 H), 1.15 (d, J ¼ 0.8 Hz, 3 H); 13C NMR (101 MHz,
CDCl3) d 163.40, 159.10, 150.70, 135.54, 134.55, 133.66, 133.36, 133.17,
130.66, 129.30, 128.59, 127.85, 127.80, 126.55, 126.47, 126.22, 125.63,
118.53, 113.77, 109.64, 88.43, 84.19, 81.41, 73.84, 71.59, 67.03, 62.23, 58.24,
55.31, 54.80, 53.63, 43.66, 41.47, 29.06, 12.47.; HRMS (ESI) calc’d for
C32H36N3O6 [M þ H]þ m/z ¼ 558.25986, found ¼ 558.26053
Nucleoside 9
Triethylamine (63 lL, 0.45 mmol) and trifluoroacetic anhydride (63 lL,
0.45 mmol) were added to a stirred solution of nucleoside 8 b (201 mg,
0.38 mmol) in a 1:1 mixture of Et2O/CH2Cl2 (2 mL) at 0ꢀC. After stirring
for 30 min, an aqueous saturated solution of NaHCO3 was added, and the
resulting mixture was extracted with CH2Cl2 (3 x 10 mL). The combined
organic extractions were dried over Na2SO4, filtered and concentrated
under reduced pressure. The residue was purified by flash chromatography
(2:8 EtOAc/hexane) to give nucleoside 9 (208 mg, 85%) as a white foam.;
1H NMR (400 MHz, CDCl3) d 7.88 – 7.77 (m, 4 H), 7.58 (d, J ¼ 0.8 Hz,
1 H), 7.55 – 7.39 (m, 5 H), 6.82 (d, J ¼ 8.7 Hz, 2 H), 5.97 (s, 1 H), 5.09
(d, J ¼ 13.6 Hz, 1 H), 4.98 (d, J ¼ 13.6 Hz, 1 H), 4.88 (d, J ¼ 11.5 Hz, 1 H),
4.79 (d, J ¼ 11.6 Hz, 1 H), 4.45 (d, J ¼ 4.0 Hz, 1 H), 4.41 (d, J ¼ 2.8 Hz,
1 H), 4.22 (dd, J ¼ 11.2, 2.6 Hz, 1 H), 3.89 – 3.79 (m, 1 H), 3.77 (s, 3 H),
3.59 (s, J ¼ 7.8 Hz, 3 H), 3.54 – 3.44 (m, 2 H), 2.20 – 2.09 (m, 1 H), 1.96 –
1.82 (m, 1 H), 1.22 (s, 3 H); 13C NMR (101 MHz, CDCl3) d 163.27,
159.16, 157.37 (d, J ¼ 36.2 Hz), 150.63, 134.72, 133.32, 133.26, 132.97,
130.73, 129.18, 128.81, 127.87, 127.84, 126.77, 126.65, 126.46, 125.50,
117.68, 113.81, 109.87, 89.17, 82.70, 79.13, 72.10, 70.94, 57.93, 55.29,
53.82, 43.73, 41.12, 29.79, 27.59, 12.62; 19F NMR (376 MHz, CDCl3) d
ꢁ69.59; HRMS (ESI) calc’d for C34H35F3N3O7 [M þ H]þ m/z ¼ 654.24216,
found ¼ 654.24315.

16
J. C. SALINAS ET AL.
Nucleoside 9a
To an stirred solution of nucleoside 9 (208 mg, 0.318 mmol) in a 9:1 mix-
ture of CH2Cl2/water (0.2 mL), 2,3-dichloro-5,6-dicyano-1,4-benzoquinone
(217 mg, 0.954 mmol) was added. The resulting mixture was stirred at
room temperature for 3 h followed by addition of aqueous 10% NaHSO3.
The resulting biphasic mixture was stirred for 10 minutes and then diluted
by addition of CH2Cl2 (10 mL). The layers were separated, then the aque-
ous layer was extracted with CH2Cl2 (3 ꢂ 10 mL). The combined organic
extractions were dried over Na2SO4, filtered and concentrated under
reduced pressure. The residue was purified by flash chromatography (4:6
Ethyl acetate/hexane) to give nucleoside 9a as a white solid (153 mg, 94%);
1H NMR (400 MHz, CDCl3) d 7.68 (s, 1 H), 7.41 (d, J ¼ 8.7 Hz, 2 H), 6.86 –
6.78 (m, 2 H), 5.93 (s, 1 H), 5.10 (d, J ¼ 13.7 Hz, 1 H), 5.01 (d, J ¼ 13.7 Hz,
1 H), 4.57 (s, 1 H), 4.45 (d, J ¼ 4.1 Hz, 1 H), 3.81 – 3.71 (m, 4 H), 3.55
(s, 3 H), 3.53 – 3.42 (m, 2 H), 2.96 (t, J ¼ 16.0 Hz, 1 H), 2.03 – 1.89 (m,
2 H), 1.87 (s, 3 H). 13C NMR (101 MHz, CDCl3) d 163.64, 159.21, 157.44
(d, J ¼ 36.0 Hz), 150.61, 133.73, 133.70, 130.63, 129.02, 116.25 (d,
J ¼ 288.2 Hz), 113.87, 109.71, 89.48, 82.73, 79.27, 63.40, 57.95, 55.34, 53.05,
43.95, 40.88, 40.84, 31.09, 13.55; 19F NMR (376 MHz, CDCl3) d ꢁ69.66;
HRMS (ESI) calc’d for C23H27F3N3O7 [M þ H]þ m/z ¼ 514.17956,
found ¼ 514.17967.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
17
Nucleoside 9 b
To an stirred solution of nucleoside 9a (153 mg, 0.297 mmol) in a 10:1 mix-
ture of MeCN:H2O (3.3 mL), Cerium ammonium nitrate (326 mg,
0.65 mmol) was added. After 24 h, water (5 mL) was added and the aqueous
layer was extracted with Et2O (3 x 10 mL). The combined organic extrac-
tions were dried over Na2SO4 and the volatiles were removed under
reduced pressure. The residue was purified by flash chromatography (1:1
1
EtOAc:hexanes) to give nucleoside 9 b (60 mg, 51%) as a white solid; H
NMR (500 MHz, CDCl3) d 9.43 (s, 1 H), 7.64 (d, J ¼ 1.3 Hz, 1 H), 5.93 (s,
1 H), 4.61 (q, J ¼ 3.6 Hz, 1 H), 4.46 (d, J ¼ 4.2 Hz, 1 H), 4.13 (dd, J ¼ 10.8,
2.3 Hz, 1 H), 3.80 – 3.73 (m, 1 H), 3.54 – 3.48 (m, 5 H), 3.18 (s, 1 H), 2.01 –
1.91 (m, 2 H), 1.85 (d, J ¼ 1.2 Hz, 3 H); 13C NMR (126 MHz, CDCl3) d
164.27, 157.52 (q, J ¼ 36.1 Hz), 150.12, 135.68, 116.25 (q, J ¼ 288.3 Hz),
110.66, 89.02, 82.73, 79.32, 63.26, 57.97, 53.15, 40.93, 30.97, 12.73; 19F
NMR (471 MHz, CDCl3) d -69.71; HRMS (ESI) calc’d for C15H19F3N3O6
[M þ H]þ m/z ¼ 394.12205, found ¼ 394.12257.
Nucleoside 10
Preparation of the levulinic anhydride solution: Levulinic acid (0.2 mL,
1.963 mmol) was added to a solution of N,N’-dicyclohexylcarbodiimide
(197 mg, 0.954 mmol) in ether (2 mL) at r.t. After 3 h, the reaction mixture
was filtered through a sintered glass keeping all the system under argon.
The remaining solid was washed with ether (3 mL) giving a solution of
Lev2O (ꢃ 0.19 M).
To a solution of nucleoside 9 b (46.6 mg, 0.118 mmol) in pyridine
(0.3 mL) was added sequentially DMAP (1 mg, 0.012 mmol) and ꢃ0.19 M
levulinic anhydride solution (1.6 mL, 0.30 mmol). After stirring for 2 h, the
volatiles were removed under reduced pressure and the solid residue was
dissolved in CH2Cl2 (30 mL). The solution was washed with sat NaHCO3
and brine. The organic phase was dried over Na2SO4 and concentrated
under reduced pressure. The residue was purified by flash chromatography
(0% MeOH to 4% CH2Cl2 using Fluorosil as stationary phase) to give
nucleoside 10 (56.2 mg, 97%) as a white foam.; 1H NMR (500 MHz,

18
J. C. SALINAS ET AL.
CDCl3) d 8.72 (s, 1 H), 7.49 – 7.45 (m, 1 H), 5.90 (s, 1 H), 5.55 (q,
J ¼ 3.5 Hz, 1 H), 4.52 (d, J ¼ 4.2 Hz, 1 H), 4.27 (dd, J ¼ 11.2, 2.9 Hz, 1 H),
3.87 – 3.80 (m, 1 H), 3.54 (d, J ¼ 0.8 Hz, 3 H), 3.51 – 3.42 (m, 2 H), 2.95 –
2.77 (m, 2 H), 2.70 – 2.51 (m, 2 H), 2.20 (s, 3 H), 2.18 – 2.02 (m, 2 H), 1.93
– 1.89 (m, 3 H); 13C NMR (126 MHz, CDCl3) d 206.46, 171.90, 163.54,
149.80, 134.62, 110.57, 89.30, 82.47, 66.42, 58.10, 54.33, 41.08, 38.13, 29.80,
28.26, 12.85; 19F NMR (471 MHz, CDCl3) d -69.68.; HRMS (ESI) calc’d for
C20H24F3N3O8 [M þ H]þ m/z ¼ 492.15883, found ¼ 492.16003.
Nucleoside 11
To a stirred solution of nucleoside 10 (56.2 mg, 0.114 mmol) in a 5:1 mix-
ture of MeOH:H2O (0.6 mL), K2CO3 (16.1 mg, 0.114 mmol) was added. The
resulting mixture was stirred at room temperature for 30 min, then silica
gel was added to the flask and the volatiles were removed under reduced
pressure. The solid residue was charged on the top of a pre-equilibrated
chromatography column and eluted with a gradient of 0% to 8% MeOH in
1
CH2Cl2 to give nucleoside 11 (31.6 mg, 70%) as a yellowish solid; H NMR
(500 MHz, CDCl3) d 7.47 (d, J ¼ 1.4 Hz, 1 H), 5.86 (s, 1 H), 5.57 (q,
J ¼ 2.9 Hz, 1 H), 3.86 (dd, J ¼ 10.8, 2.7 Hz, 1 H), 3.75 (d, J ¼ 4.8 Hz, 1 H),
3.57 (s, 3 H), 3.11 – 3.00 (m, 2 H), 2.93 (td, J ¼ 13.4, 3.1 Hz, 1 H), 2.88 –
2.75 (m, 2 H), 2.68 (ddd, J ¼ 16.9, 7.7, 5.1 Hz, 1 H), 2.59 (ddd, J ¼ 16.9, 6.9,
5.4 Hz, 1 H), 2.19 (s, 3 H), 2.02 (ddq, J ¼ 13.7, 3.6, 1.7 Hz, 2 H), 1.91 (d,
J ¼ 1.2 Hz, 3 H), 1.66 – 1.56 (m, 1 H); 13C NMR (126 MHz, CDCl3) d
206.39, 171.95, 164.07, 150.14, 135.12, 110.48, 88.47, 83.69, 79.22, 69.25,
58.34, 55.56, 41.29, 38.02, 29.88, 29.78, 28.35, 12.75; HRMS (ESI) calc’d for
C18H25N3O7 [M þ H]þ m/z ¼ 396.17653, found ¼ 396.17645.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
19
Nucleoside 11a
To a stirred solution of nucleoside 11 (21.6 mg, 0.0546 mmol) and H-
phosphonate 14 (40 mg, 0.071 mmol) in 1:1:1 mixture of
a
CCl4:MeCN:CH2Cl2 (2.1 mL), triethylamine (10 lL, 0.071 mmol) was added.
After 3 h, the volatiles were removed under reduced pressure and the dry
residue was purified by flash chromatography (100% EtOAc) to give
nucleoside 11a (42.6 mg, 82%) as a ꢃ 1:1 mixture of P-isomers (31P NMR);
1H NMR (400 MHz, CDCl3) d 9.59 (s, 1 H), 9.10 (d, J ¼ 14.1 Hz, 1 H), 7.65
– 7.58 (m, 4 H), 7.49 – 7.30 (m, 8 H), 6.46 – 6.34 (m, 1 H), 5.82 (d,
J ¼ 4.3 Hz, 1 H), 5.39 (d, J ¼ 52.2 Hz, 1 H), 4.40 – 4.26 (m, 1 H), 4.22 – 3.97
(m, 4 H), 3.95 – 3.79 (m, 1 H), 3.73 – 3.55 (m, 5 H), 3.55 – 3.42 (m, 2 H),
3.08 – 2.74 (m, 5 H), 2.69 – 2.48 (m, 2 H), 2.33 (ddd, J ¼ 13.4, 5.6, 2.1 Hz,
1 H), 2.18 (d, J ¼ 8.3 Hz, 3 H), 2.04 (s, 1 H), 1.92 – 1.79 (m, 7 H), 1.07 (d,
J ¼ 3.4 Hz, 9 H); 13C NMR (101 MHz, CDCl3) d 206.44, 206.43, 171.99,
171.87, 164.08, 163.97, 163.83, 150.40, 150.08, 135.85, 135.85, 135.81,
135.68, 135.33, 134.67, 134.57, 133.14, 133.11, 133.02, 132.90, 130.36,
130.33, 130.25, 128.16, 128.13, 128.08, 128.06, 111.25, 110.98, 110.13,
110.09, 88.55, 88.50, 85.94, 85.85, 85.70, 85.63, 85.41, 85.39, 82.72, 78.52,
78.40, 74.05, 73.24, 67.64, 67.58, 60.52, 57.54, 57.45, 54.23, 54.22, 54.22,
53.48, 53.43, 53.43, 53.22, 53.17, 41.03, 41.01, 40.93, 40.90, 40.71, 38.07,
29.81, 29.77, 28.30, 26.96, 26.91, 21.17, 19.13, 19.10, 14.32, 12.84, 12.52,
12.40; 31P NMR (162 MHz, CDCl3) d 8.38, 7.73; HRMS (ESI) calc’d for
C45H58N5O14PSi [M þ H]þ m/z ¼ 952.35599, found ¼ 952.35676.
Nucleoside 12
To a stirred solution of nucleoside 11a (36 mg, 0.038 mmol) in THF
(2.0 mL), tetrabutylammonium fluoride (1 M, 46 lL, 0.045 mmol) in THF
was added. After 20 min, solid silica gel was added to the reaction mixture
and the volatiles were removed under reduced pressure. The solid residue
was charged in on the top of a pre-equilibrated chromatography column
and eluted with a gradient of 0% to 8% MeOH in CH2Cl2, to give nucleo-
1
side 12 (25 mg, 92%) as a ꢃ 1:1 mixture of P-isomers (31P NMR). H NMR
(300 MHz, CDCl3) d 10.09 – 9.48 (m, 2 H), 7.56 – 7.29 (m, 2 H), 6.23 (dt,

20
J. C. SALINAS ET AL.
J ¼ 18.1, 6.5 Hz, 1 H), 5.82 (d, J ¼ 4.3 Hz, 1 H), 5.48 (s, 1 H), 4.67 – 4.56 (m,
1 H), 4.53 – 4.05 (m, 6 H), 3.75 (d, J ¼ 11.3 Hz, 3 H), 3.63 (d, J ¼ 6.0 Hz,
3 H), 3.39 – 3.24 (m, 3 H), 3.19 – 2.99 (m, 2 H), 2.95 – 2.79 (m, 2 H), 2.72
– 2.52 (m, 2 H), 2.44 – 2.32 (m, 1 H), 2.19 (d, J ¼ 1.2 Hz, 3 H), 1.93 – 1.85
(m, 6 H), 1.44 (h, J ¼ 7.3 Hz, 2 H); 13C NMR (101 MHz, CDCl3) d 206.76,
206.63, 172.00, 171.92, 164.60, 164.19, 164.16, 164.09, 150.62, 150.60,
150.51, 150.28, 135.82, 135.53, 135.06, 134.73, 111.19, 111.15, 110.22,
109.94, 88.71, 88.47, 85.26, 85.15, 85.09, 85.02, 84.94, 82.61, 82.47, 78.75,
78.63, 78.50, 71.25, 70.63, 67.73, 66.54, 66.48, 65.89, 65.84, 60.52, 59.13,
57.58, 57.49, 54.41, 54.20, 54.18, 53.76, 53.71, 53.62, 53.57, 41.45, 41.11,
40.28, 39.98, 38.10, 29.83, 28.36, 24.19, 19.88, 13.78, 12.87, 12.84, 12.64,
12.52; 31P NMR (121 MHz, CDCl3) d 9.23, 8.51; HRMS (ESI) calc’d for
C29H40N5O14P [M þ H]þ m/z ¼ 714.23821, found ¼ 714.23811.
H-phosphonate 14
To a solution of nucleoside 13 (0.26 mg, 0.53 mmol) and diisopropylethyl-
amine (0.38 mL, 2.14 mmol) at 0ꢀC was added N,N-diisopropylmethylchlor-
ophosphoramidite (0.21 mL, 1.1 mmol). The resulting solution was stirred
at rt for 30 min, cooled to 0ꢀC, then diluted with EtOAc, washed with cold
10% sodium bicarbonate, dried over Na2SO4, and concentrated under
reduced pressure. The residue was purified by flash chromatography (1:2
EtOAc;hexanes þ 1% NEt3) to give phosphoramidite 13a (0.29 mg, 97%).
To a solution of phosphoramidite 13a (0.29 mg, 0.45 mmol) in MeCN
(4 mL), a 0.45 M solution of 1 H-tetrazole (3 mL, 1.36 mmol) and water
(0.8 mL) were added. After 30 min, the reaction mixture was diluted with
EtOAc (50 mL) and washed sequentially with water, 5% NaHCO3, and
brine. The organic layer was dried over Na2SO4, and concentrated under
reduced pressure. The residue was purified by flash chromatography
(EtOAc) to givephosphoramidate 14 (0.23 mg, 91%) as a 1:1 mixture of
1
compounds (31P NMR); H NMR (400 MHz, CDCl3) d 9.53 (s, 1 H), 7.68 –
7.58 (m, 4 H), 7.55 – 7.50 and 5.78 – 5.73 (H-P, m, 1 H), 7.48 – 7.34 (m,
6 H), 7.31 – 7.24 (m, 2 H), 6.49 – 6.41 (m, 1 H), 4.41 – 4.30 (m, 1 H), 4.08
– 4.01 (m, 1 H), 3.97 – 3.87 (m, 1 H), 3.72 – 3.56 (m, 4 H), 2.39 – 2.30 (m,

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
21
1 H), 1.95 – 1.83 (m, 4 H), 1.07 (s, 9 H).; 13C NMR (101 MHz, CDCl3) d
163.97, 150.56, 135.76, 135.72, 135.27, 135.22, 133.02, 132.97, 132.75,
132.73, 130.33, 130.30, 130.26, 128.08, 128.06, 128.05, 111.47, 85.24, 85.17,
84.88, 84.81, 72.89, 72.72, 64.75, 64.70, 64.63, 64.57, 52.30, 52.24, 52.18,
52.13, 40.56, 40.49, 26.89, 19.03, 12.41; 31P NMR (162 MHz, CDCl3) d 10.10,
9.05; HRMS (ESI) calc’d for C27H36N2O7PSi [M þ H]þ m/z ¼ 559.20239,
found ¼ 559.20271.
Nucleoside dimer 16
2-Cyanoethyl tetraisopropylphosphordiamidite (0.084 mmol, 0.025 g,
0.027 mL) was added to a suspension of the nucleoside dimer 12
(0.035 mmol, 0.025 g) and diisopropylammonium tetrazolide (0.011 mmol,
0.0018 g) in dry acetonitrile (0.19 mL). The reaction was stirred at room
temperature for 4 hours at which time analysis of the reaction by LCMS
showed conversion to 15 (calc’d for C38H56N7O15P2, [M-H]ꢁ m/z ¼ 912.3,
found 912.3). The reaction was diluted with ethyl acetate and loaded on
silica gel prewashed with 50% THF/3% triethylamine/ethyl acetate. The
product was eluted from the column using 70% THF/3% triethylamine/
ethyl acetate and concentrated under reduce pressure to yield 16 (0.012 g,
36%) as a white foam (calc’d for C38H56N7O16P2, [M-H]ꢁ m/z ¼ 928.3,
found 928.3). 31P NMR (121 MHz, CDCl3) d 8.92, 8.15, 7.95, 7.83.
ORCID
Punit P. Seth
http://orcid.org/0000-0002-1783-7806
References
[1] Wan, W. B.; Seth, P. P. The Medicinal Chemistry of Therapeutic Oligonucleotides.
J. Med. Chem. 2016, 59, 9645. DOI: 10.1021/acs.jmedchem.6b00551.
[2] Shen, X.; Corey, D. R. Chemistry, Mechanism and Clinical Status of Antisense
Oligonucleotides and Duplex RNAs. Nucleic Acids Res. 2018, 46, 1584. DOI: 10.1093/
nar/gkx1239.

22
J. C. SALINAS ET AL.
[3] Beierlein, J. M.; McNamee, L. M.; Ledley, F. D. As Technologies for Nucleotide
Therapeutics Mature, Products Emerge. Mol. Ther. Nucleic Acids. 2017, 9, 379. DOI:
10.1016/j.omtn.2017.10.017.
[4] Obika, S.; Nanbu, D.; Hari, Y.; Morio, K-i.; In, Y.; Ishida, T.; Imanishi, T. Synthesis
of 20-O,40-C-Methyleneuridine and -Cytidine. Novel Bicyclic Nucleosides Having
a Fixed C3, -Endo Sugar Puckering. Tetrahedron Lett 1997, 38, 8735. DOI: 10.1016/
S0040-4039(97)10322-7.
[5] Imanishi, T.; Obika, S. BNAs: novel Nucleic Acid Analogs with a Bridged Sugar
Moiety. Chem. Commun. 2002, 1653. DOI: 10.1039/b201557a.
[6] Rajwanshi, V. K.; Håkansson, A. E.; Sørensen, M. D.; Pitsch, S.; Singh, S. K.; Kumar,
R.; Nielsen, P.; Wengel, J. The Eight Stereoisomers of LNA (Locked Nucleic Acid):
a Remarkable Family of Strong RNA Binding Molecules. Angewandte Chemie 2000,
112, 1722. DOI: 10.1002/(SICI)1521-3757(20000502)112:9<1722::AID-ANGE1722>3.
0.CO;2-Z.
[7] Giacometti, R. D.; Salinas, J. C.; Ostergaard, M. E.; Swayze, E. E.; Seth, P. P.;
Hanessian, S. Design, Synthesis, and duplex-stabilizing properties of conformation-
ally constrained tricyclic analogues of LNA. Org. Biomol. Chem. 2016, 14, 2034.
DOI: 10.1039/c5ob02576a.
[8] Hanessian, S.; Schroeder, B. R.; Giacometti, R. D.; Merner, B. L.; Østergaard, M.;
Swayze, E. E.; Seth, P. P. Structure-based design of a highly constrained nucleic acid
analogue: improved duplex stabilization by restricting sugar pucker and torsion
angle c. Angew. Chem. Int. Ed. Engl. 2012, 51, 11242. DOI: 10.1002/anie.201203680.
ꢀ
[9] Dupouy, C.; Iche-Tarrat, N.; Durrieu, M.-P.; Rodriguez, F.; Escudier, J.-M.; Vigroux,
A. Watson-Crick base-pairing properties of nucleic acid analogues with stereocon-
trolled alpha and beta torsion angles (alpha,beta-D-CNAs) ). Angew. Chem. Int. Ed.
Engl. 2006, 45, 3623. DOI: 10.1002/anie.200504475.
[10] Salinas, J. C.; Yu, J.; Østergaard, M.; Seth, P. P.; Hanessian, S. Conception and
Synthesis of Oxabicyclic Nucleoside Phosphonates as Internucleotidic Phosphate
Surrogates in Antisense Oligonucleotide Constructs. Org. Lett. 2018, 20, 5296. DOI:
10.1021/acs.orglett.8b02233.
[11] Wang, G. Conformationally Locked Nucleosides. Synthesis and Stereochemical
Assignments of 30-N,50-C-Bridged 30-Amino-30-Deoxythymidines. Tetrahedron Lett
1999, 40, 6343. DOI: 10.1016/S0040-4039(99)01205-8.
[12] Wang, G.; Stoisavljevic, V. Conformationally Locked Nucleosides. Synthesis of
Oligodeoxynucleotides
Containing
30-amino-30-deoxy-30-N,50(R)-C-ethylene-
thymidine. Nucleosides. Nucleotides Nucleic Acids. 2000, 19, 1413. DOI: 10.1080/
15257770008033851.
[13] Garcia Fernandez, J. M.; Ortiz Mellet, C.; Jimenez Blanco, J. L.; Fuentes, J. Enantiopure
2-Thioxotetrahydro-1,3-O,N-Heterocycles from Carbohydrates. 3. Enantiopure C-4
Chiral Oxazine- and Oxazolidine-2-Thiones from 3-Deoxy-3-Isothiocyanato Sugars.
J. Org. Chem. 1994, 59, 5565. DOI: 10.1021/jo00098a014.
ꢀ
[14] Gruner, S. A. W.; Keri, G.; Schwab, R.; Venetianer, A.; Kessler, H. Sugar Amino
Acid Containing Somatostatin Analogues That Induce Apoptosis in Both Drug-Sensitive
and Multidrug-Resistant Tumor Cells. Org. Lett 2001, 3, 3723.
[15] Gateau-Olesker, A.; Castellanos, L.; Panne-Jacolot, F.; Cleophax, J.; Gero, S. D.
ꢀ
ꢀ
ꢀ ꢀ
Ouverture Regiospecifique D’un Oxirane Prepare a Partir du d-Xylose Par Des
Carbanions Soufres. Tetrahedron 1981, 37, 1685. DOI: 10.1016/S0040-4020(01)98931-8.
ꢀ
ꢀ
ꢁ
[16] Cristobal Lopez, J.; Lameignere, E.; Burnouf, C.; de los Angeles Laborde, M.; Ghini,
A. A.; Olesker, A.; Lukacs, G. Efficient Routes to Pyranosidic Homologated

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
23
Conjugated Enals and Dienes from Monosaccharides. Tetrahedron 1993, 49, 7701.
DOI: 10.1016/S0040-4020(01)87245-8.
[17] Bernotas, R. C.; Cube, R. V. The Use of Triphenylphosphine/Diethyl
Azodicarboxylate (Dead) for the Cyclization of 1,4- and 1,5-Amino Alcohols.
Tetrahedron Lett 1991, 32, 161. DOI: 10.1016/0040-4039(91)80843-U.
[18] Gommermann, N.; Knochel, P. Practical Highly Enantioselective Synthesis of
Terminal Propargylamines. An Expeditious Synthesis of (S)-(þ)-Coniine. Chem.
Commun. 2004, 2324. DOI: 10.1039/b409951f.
[19] Wekesa, F. S.; Phadke, N.; Jahier, C.; Cordes, D. B.; Findlater, M. A Simple and
Convenient Method for the Synthesis of N,N-Diaryl Tertiary Amines. Synthesis
2014, 46, 1046. DOI: 10.1055/s-0033-1340820.
[20] Kumar, K. S. A.; Rathee, J. S.; Subramanian, M.; Chattopadhyay, S. Divergent
Synthesis of 4-epi-fagomine, 3,4-dihydroxypipecolic acid, and a dihydroxyindolizidine
and their b-galactosidase inhibitory and immunomodulatory activities. J. Org. Chem.
2013, 78, 7406. DOI: 10.1021/jo400448p.
[21] Niedballa, U.; Vorbru€ggen, H. A General Synthesis of Pyrimidine nucleosides.
Angew. Chem. Int. Ed. Engl. 1970, 9, 461. DOI: 10.1002/anie.197004611.
[22] Prudhomme, D. R.; Park, M.; Wang, Z.; Buck, J. R.; Rizzo, C. J. Synthesis of 20
Deoxyribonucleosides: b-30,50-di-o-Benzoylthymidine. Org. Synth 2000, 77, 162.
€
[23] Bleicher, K. H.; Wuthrich, Y.; Adam, G.; Hoffmann, T.; Sleight, A. J. Parallel
Solution- and Solid-Phase Synthesis of Spiropyrrolo-Pyrroles as Novel Neurokinin
Receptor Ligands. Bioorg. Med. Chem. Lett 2002, 12, 3073.
[24] Madsen, A. S.; Jørgensen, A. S.; Jensen, T. B.; Wengel, J. Large Scale Synthesis of
20-amino-LNA Thymine and 5-methylcytosine nucleosides. J. Org. Chem. 2012, 77,
10718. DOI: 10.1021/jo302036h.
[25] Prakash, T. P.; Lima, W. F.; Murray, H. M.; Li, W.; Kinberger, G. A.; Chappell,
A. E.; Gaus, H.; Seth, P. P.; Bhat, B.; Crooke, S. T.; Swayze, E. E. Identification
of Metabolically Stable 5’-phosphate analogs that support single-stranded siRNA
activity. Nucleic Acids Res. 2015, 43, 2993. DOI: 10.1093/nar/gkv162.
[26] A.; Gentle, C.Structure–Function Studies on Nucleoside Antibiotic Mureidomycin A:
synthesis of 50-Functionalised Uridine Models. J. Chem. Soc, Perkin Trans. 1 1999,
1287.; A.; Harrison, S.; Inukai, M.; D. H. Bugg, T. DOI: 10.1039/a901287g.
[27] Macchione, G.; Maza, S.; Mar Kayser, M.; de Paz, J. L.; Nieto, P. M. Synthesis
of Chondroitin Sulfate Oligosaccharides Using N-(Tetrachlorophthaloyl)- and
N-(Trifluoroacetyl)Galactosamine Building Blocks. Eur. J. Org. Chem. 2014, 2014,
3868. DOI: 10.1002/ejoc.201402222.
[28] Lan, T.; McLaughlin, L. W. Minor Groove Hydration Is Critical to the Stability of
DNA Duplexes. J. Am. Chem. Soc. 2000, 122, 6512. DOI: 10.1021/ja000686v.