Large scale synthesis of 2′-Amino-LNA thymine and 5-methylcytosine nucleosides

Article abstract of DOI:10.1021/jo302036h

Thymine intermediate 17 has been synthesized on a multigram scale (50 g, 70 mmol) from starting sugar 1 in 15 steps in an overall yield of 73%, with only 5 purification steps. The key thymine intermediate 18 was obtained from 17 in a single step in 96% yi

Full text of DOI:10.1021/jo302036h

Article
pubs.acs.org/joc
Large Scale Synthesis of 2′-Amino-LNA Thymine and
5‑Methylcytosine Nucleosides
Andreas Stahl Madsen,† Anna Søndergaard Jørgensen, Troels Bundgaard Jensen, and Jesper Wengel*
Nucleic Acid Center, Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, 5230 Odense M, Denmark
S
* Supporting Information
ABSTRACT: Thymine intermediate 17 has been synthesized on a multigram scale (50 g, 70 mmol) from starting sugar 1 in 15
steps in an overall yield of 73%, with only 5 purification steps. The key thymine intermediate 18 was obtained from 17 in a single
step in 96% yield, whereas the key 5-methylcytosine intermediate 20 was obtained from 17 in 2 steps in 58% yield. This highly
efficient large scale route necessitates only 2 and 3 novel steps to obtain N2′-functionalized thymine and 5-methylcytosine
amino-LNA phosphoramidites from these key intermediates, respectively.
perylene derivatives.28,34−41 Remarkably, these functionalities
generally could be introduced without compromising the high
thermal stability typical of LNA-containing duplexes. This is in
INTRODUCTION
■
Incorporation of functionalized nucleosides into oligonucleo-
tides (ONs) offers the opportunity of site specific conjugation
of functional entities to nucleic acid structures. An enormous
body of functionalized nucleotides has been synthesized and
incorporated into ONs, including functionalization of the
nucleobase with fluorescent moieties (recent examples include
contrast to related 2′-amino-uridine derivatives, where N2′-
functionalization usually leads to significantly lower duplex
thermal affinity.31
The first reports on 2′-amino-LNA nucleotides as con-
C5 of pyrimidines,1−6 C8 of purines,7,8 and C7 of 7-
jugation site used synthetic strategies that were either not
deazapurines6,9,10), substitution of the nucleobase with
fluorescent moieties,11−14 functionalization of the sugar
moiety,15−17 and functionalization of the phosphodiester
moiety.18−21 Non-nucleosidic structures have also been used
to position functional groups in a duplex context, including
forced intercalation probes (FIT, thiazole-orange modified
PNAs),22,23 intercalating nucleic acid (INA),24,25 or twisted
intercalating acid (TINA).26,27
2′-Amino-LNA compares favorably with other strategies for
defined positioning of functional moieties in the duplex, by the
unique combination of conformational restriction and a
precisely positioned conjugation point. The bicyclic skeleton
of 2′-amino-LNA leads to (a) a locked 3′-endo furanose
conformation, (b) improved binding toward DNA and RNA
complementary strands, and (c) pronounced mismatch
discrimination. Functionalities that have successfully been
introduced in the minor groove of duplexes via the N2′-atom
of 2′-amino-LNA nucleotides include alkyl and acyl moi-
eties,28,29 metal chelators,30−32 polar moieties like nucleo-
bases33 and amino acids,29 and aromatic groups like pyrene and
compatible with introduction of a wide variety of N2′-
functionalities,42 or demanded several steps for each introduced
functionality.28 Later syntheses have, however, used a slightly
different strategy, where key intermediate 18 is first N2′-
functionalized and then O3′-phosphitylated. This strategy thus
necessitates only two novel steps to obtain a phosphoramidite
derivative of each N2′-functionality (see Figure 1).
In our continued investigation of functionalized 2′-amino-
LNA, we therefore wanted to establish a reliable and efficient
large scale synthesis of key intermediate 18. While the smaller
scale synthesis of 3′-O-benzyl-2′-amino-LNA thymine deriva-
tive 15 has been described,43 a large scale synthesis has never
been reported. Furthermore, we wanted to amend the fact that
the synthesis of amino alcohol 18 never has been reported. The
majority of functionalized 2′-amino-LNA derivatives have been
synthesized with thymine as the nucleobase, but we further
wanted to pursue synthesis of a key 5-methylcytosine
Received: September 21, 2012
Published: November 12, 2012
© 2012 American Chemical Society
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Figure 1. Synthesis of functionalized 2′-amino-LNA thymine. a) either aldehyde and NaBH(OAc)3 or carboxylic acid and coupling reagent (e.g.,
HATU or EDC·HCl), (b) 2-cyanoethyl N,N-diisopropylphosphoramidochlorodite and diisopropylethylamine or bis(N,N-diisopropylamino)-2-
cyanoethoxyphosphine and diisopropylammonium tetrazolide, c) DNA synthesis. See text for references to specific examples.
a
Scheme 1
a
(a) MsCl, pyridine, 0 °C to rt, 20 h; (b) 80% aq F3CCO2H, 0 °C, 4 h; (c) Ac2O, pyridine, 0 °C to rt, 40 h; (d) thymine, N,O-
bis(trimethylsilyl)acetamide, TMSOTf, MeCN, 60 °C, 96 h; (e) sat. NH3 in MeOH, 0 °C, 4.5 h; (f) MsCl, pyridine/DCM (50:50, v/v), 0 °C, 5 h;
(g) DBU, MeCN, 0 °C to rt, 18 h (86% from 1); (h) H2SO4 (0.5 M), H2O/acetone (50:50, v/v), reflux, 3 h (98%).
intermediate, thereby expanding the potential sequence context
and applicability of functionalized 2′-amino-LNA oligonucleo-
tides.
isolated after precipitation in 86% yield (calculated from
starting sugar 1, Scheme 1). This intermediate was
subsequently converted to β-D-threo-configured nucleoside 9
in 98% yield by acidic hydrolysis. A more concentrated reaction
mixture (0.5 M H2SO4 in H2O/acetone (50:50, v/v) compared
to 0.05 M H2SO4 in H2O/acetone (50:50, v/v) previously
reported) was more easy to handle on large scale and allowed a
reduction in reaction time without compromising the yield.
To our surprise, the two step procedure of activation of the
2′-hydroxy group by 2 equiv of triflic anhydride, 4 equiv of
DMAP in pyridine and dichloromethane (affording triflate 10)
followed by azidation using sodium azide in DMF did not
afford the desired azide 11 in the high yields previously
reported for a smaller scale transformation.43 Instead, azide 11
was formed only in modest yields (∼50%) along with
formation of diazide 12 in >20% yield (Figure 2).
RESULTS AND DISCUSSION
■
Key Intermediate 17 and Thymine Derivative 18.
Starting from known 4′-C-hydroxymethyl ribose derivative 1,44
mesylation, isopropylidene cleavage and acetylation was carried
out on 275 mmol scale, to afford coupling sugar 4 without the
need of purification. The two step procedure of isopropylidene
cleavage using aqueous trifluoroacetic acid followed by
acetylation using acetic anhydride in pyridine was chosen
over the one step procedure of acetic anhydride in glacial acetic
acid and catalytic amounts of sulfuric acid,45 since the former
conditions led to less colorization of the crude product.
Subsequent introduction of the nucleobase was carried out on
Presumably, the reaction conditions employed during the
first of the two steps mentioned above leads to activation of the
4-OH enol tautomer of the nucleobase as well as the 2′-hydroxy
group, allowing incorporation of two azido moieties. This is in
line with the observation that thymine nucleosides can be
activated by phosphorus oxychloride, as known from the
widespread thymine to 5-methylcytosine, or uracil to cytosine,
conversion. Furthermore, it is known that activation of 2-
pyrimidones by 4-chlorophenylphosphorodichloridate in the
presence of pyridine lead to formation of fluorescent
100 mmol scale using the modified Vorbruggen coupling
̈
conditions recently described for coupling of uracil to coupling
sugar 4.46 The reduced temperature (60 °C compared to reflux
(82 °C)) led to longer reaction time, but afforded nucleoside 5
in a purity sufficient to proceed without the need for
purification. Nucleoside 5 was subsequently deacetylated in
saturated methanolic ammonia affording alcohol 6 without any
detectable ring formation between the O2′-atom and the C4′-
hydroxymethyl group. Mesylation and treatment with anhy-
drous DBU afforded 2,2′-O-anhydro compound 8, which was
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Figure 2. Formation of azides 11 and 12 from alcohol 9 under suboptimal conditions.
a
Scheme 2
a
(a) Tf2O, DMAP, pyridine, DCM, −70 to 0 °C, 4 h (97%); (b) NaN3, DMF, rt, 18 h; (c) PMe3, NaOH, THF, H2O, 0 °C to rt, 7 h; (d) KOBz, 18-
crown-6, dioxane, 80 °C, 33 h (94% from 10); (e) satd NH3/MeOH, 0 °C to rt, 36 h; (f) (CF3CO)2O, pyridine/DCM (50:50, v/v), −70 to 0 °C, 30
min; (g) DMTCl, pyridine/DCM (50:50, v/v), rt, 18 h (95% from 14); (h) NH4HCO2, 20% Pd(OH)2/C, MeOH, reflux, 3 h (96%).
pyrimidinyl-pyridinium salts which can then serve as inter-
mediates for the synthesis of various 4-substituted 2-
pyrimidinones including 4-azido-2-pyrimidones.47 In agreement
with these results, a very polar fluorescent compound could be
identified by TLC (as evaluated by irradiation at 365 nm). The
fluorescence of the unwanted intermediate allowed us to
qualitatively evaluate the amount formed under various
activation conditions (1.3−2.0 equiv of Tf2O, 0.05−4.0 equiv
of DMAP and 0−10 equiv of pyridine). In our hands, we found
that 1.4 equiv of triflic anhydride, 2 equiv of DMAP and 10
equiv of pyridine and adding the anhydride at −70 °C resulted
in almost no formation of the fluorescent compound.
Surprisingly, aqueous workup of the reaction mixture after
the first step did not efficiently remove the polar fluorescent
compound. However, column chromatography using ethyl
acetate as eluent resulted in easy removal of any polar
intermediates and isolation of the surprisingly stable triflate
10 in 97% yield on 85 mmol scale, thereby omitting the need
for cumbersome separation of azides 11 and 12 (Scheme 2).
Azidation, Staudinger reduction under basic conditions to
ensure immediate ring closure, and exchange of the 5′-mesyloxy
group to benzoate afforded nucleoside 14 in 94% yield on a 80
mmol scale from triflate 10 without the need of purification of
intermediates. The use of a more concentrated reaction
solution than previously reported in the azidation step and
the use of dioxane as solvent rather than DMF in the last step
simplified the large scale workup procedures significantly.
Ammonolysis of the benzoate ester afforded amino alcohol 15,
which could be reacted with trifluoroacetic anhydride in
pyridine and dichloromethane to give N2′-trifluoroacetyl
derivative 16 followed by dimethoxytritylation to afford fully
protected nucleoside 17 in 95% yield from nucleoside 14 on 75
mmol scale. Finally, debenzylation by transfer hydrogenation
using ammonium formate as hydrogen source and Pd(OH)2/C
as catalyst on a 26 mmol scale, also resulting in removal of the
N2′-trifluoroacetyl group, furnished the desired key inter-
mediate 18 in 96% yield. This corresponds to an overall yield of
70% for the 16 step conversion 1→18 involving only six
purification procedures.
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a
Scheme 3.
a
(a) (i) POCl3, 1,2,4-triazole, NEt3, MeCN, 0 °C to rt, 75 min, (ii) NH3, H2O, THF, rt, 70 min (quantitative); (b) NH4HCO2, 20% Pd(OH)2/C,
MeOH, reflux, 3 h (58%); (c) 21a: BzCl, pyridine, rt 24 h (74%) or 21b: Ac2O, DMAP, pyridine/DCM (50:50, v/v), rt, 5 days (82%.
5-Methylcytosine Derivatives. While thymine derivative
18 is highly useful as divergence point in the synthesis of
functionalized amino-LNA thymine derivative, synthesis of 5-
methylcytosine derivatives are slightly more demanding, owing
to N4-protection which is necessary for oligonucleotide
synthesis. A single N2′-functionalized 5-methylcytosine de-
rivative has previously been prepared and incorporated into
ONs by Hrdlicka and co-workers.48 Initially following the same
route, key intermediate 17 was conveniently and in quantitative
yield converted to 5-methylcytosine derivative 19 on a 7 mmol
scale via the well-established two-step triazolo/ammonolysis
methodology.49 Thymine derivative 17 was thus treated with
POCl3, 1,2,4-triazole and Et3N to afford the 4-triazolothymine
derivative, which was converted to the corresponding 5-
methylcytosine derivative 19 by treatment with aqueous
ammonia in THF (Scheme 3).
Debenzylation and trifluoroacetamide cleavage by transfer
hydrogenation using ammonium formate as hydrogen source
and Pd(OH)2/C as catalyst, then afforded diamino alcohol 20
in 58% yield. We did not pursue isolation and identification of
byproducts; however, the relatively low yield of 20 probably
reflects several aspects. While small scale conversion of thymine
derivative 17 to 5-methylcytosine derivative 19 proceeded
smoothly, upscaling to 25 mmol scale and the use of crude 19
in the subsequent hydrogenation reaction (concomitant
debenzylation and trifluoroacetamide cleavage) might affect
yields. Furthermore, purification of the very polar diamino
alcohol 20 was severely complicated by residual ammonium
formate, which might also negatively affect the isolated yield.
Interestingly, crude diamino alcohol 20 was insoluble in DCM,
whereas purified diamino alcohol 20 was readily soluble. We
hypothesize that crude compound 20 precipitated as a formate
salt, whereas the laborious workup broke the salt formation,
leaving diamino alcohol 20 after purification. In summary, in
view of the four-step procedure with no intermediate
purifications and a final purification of the highly polar product,
compound 20 is obtained from key intermediate 17 in an
acceptable yield. This key intermediate is set up for a three step
sequence of N2′-functionalization, N4-protection and O3′-
phosphitylation to afford phosphoramidite building blocks
ready for solid phase oligonucleotides synthesis.
As a consequence, three steps are needed for each N2′-
functionalization of the 5-methylcytosine derivative. Alternative
routes that could reduce the number of diverged steps were
therefore explored. 5-Methylcytosine derivative 19 is well-
suited for instalment of an appropriate 4-amino protection
group (e.g., benzoyl or acetyl). Both protection groups were
easily introduced affording acylated nucleosides 21a and 21b.
Subsequent debenzylation, however, proved problematic;
hydrogenation under a H2 atmosphere using Pd/C or
Pd(OH)2/C as catalyst gave a complex mixture of products,
whereas in situ hydrogen generation using cyclohexene as
hydrogen source and either Pd/C or Pd(OH)2/C as catalyst
led to O5′-detritylation while leaving the O3′-benzyl group
intact. Transfer hydrogenation using ammonium formate as
hydrogen source and Pd/C as catalyst resulted in debenzylation
but with concomitant removal of the 2′-N- and 4-N-acyl
groups, affording diamino alcohol 20, where to a more efficient
synthesis was established above. Furthermore, selective N4-
benzoylation of diamino alcohol 20 was attempted by a
transient protection strategy using two equivalents of TMSCl
followed by addition of BzCl. This procedure however resulted
in N2′-benzoylation in agreement with the expected higher
nucleophilicity of the secondary aliphatic 2′-C-amino group
over the exocyclic 4-C-amino group. These results are in
agreement with the previous report, where selective N4-
protection also failed.48 Alternative routes, including instalment
of an orthogonal N2′-protection group prior to N4-acylation,
followed by N2′-deprotection to allow a two-step divergent
strategy, as used by Hrdlicka and co-workers, could be
envisioned. However, since the N2′-/N4-selectivity of such
protection group would not be expected to be superior to direct
instalment of the desired functionality, we conclude, also in
light of the extra synthetic and purification steps needed, that
the three-step diverged strategy (N2′-functionalization, N4-
protection and O3′-phosphitylation) most likely is the most
viable route toward N2′-functionalized 5-methylcytosine
amino-LNA phosphoramidites. This is corroborated by the
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spectra) and follow standard nucleoside nomenclature. Systematic
compound names are given according to von Baeyer nomenclature.
High resolution mass spectra (HRMS) were performed on a TOF
(time-of-flight) analyzer and were recorded in positive ion mode using
electrospray ionization (ESI).
results obtained by Hrdlicka and co-workers, where the
trifluoroacetyl group serves as orthogonal N2′-protection
group, affording the N4-benzoylated derivative in 30% yield
from 20, followed by N2′-functionalization and phosphityla-
tion. To confirm the practicability of the three-step route, N2′-
palmitoylation of 5-methylcytosine intermediate 20, followed
by N4-benzoylation and O3′-phosphitylation was used to afford
phosphoramidite 23. Reaction scheme can be found in the
Supporting Information. In agreement with the expected N2′-
selectivity of 5-methylcytosine derivative 20, acylation gave
predominantly the N2′-palmitoylated intermediate, which upon
benzoylation afforded N2′-palmitoyl,4-N-benzoyl intermediate
22 in 45% yield from diamino alcohol 20. The relatively low
yield of intermediate 22 is likely a consequence of some
dipalmitoylation at N2′ and 4-N in the first step. In agreement
herewith, formation of a less polar compound was observed,
but not further analyzed. While selectivity of the first step might
be improved, phosphoramidite 23 was obtained in 38% yield
from intermediate 20, thereby demonstrating the feasibility of
the three-step route.
1,2-Di-O-acetyl-3-O-benzyl-4-C-methanesulfonoxymethyl-5-O-
methanesulfonyl-D-erythro-pentofuranose (4).50 Two reaction
batches (274 mmol each) were used for the steps 1→4, a
representative protocol is given here: Diol 1 (85.0 g, 274 mmol)
was coevaporated with pyridine (100 and 50 mL) and then dissolved
in anhydrous pyridine (430 mL) and cooled to 0 °C. MsCl (53 mL,
685 mmol) was added dropwise over 20 min. The reaction mixture
was stirred for 20 h, during which the reaction was allowed to heat to
rt and the clear reaction mixture turned first green and then brown
upon stirring. Ice (400 mL) was added and the reaction mixture was
extracted with EtOAc (400 mL and 2 × 200 mL). The org. phase was
dried over MgSO4, and evaporated to dryness to afford dimesylate 2 as
a brownish syrup which was used directly in the next step. TLC (50%
EtOAc in PE, v/v): Rf (1) = 0.3, Rf (2) = 0.5. Crude isopropylidene
derivative 2 (128 g) was suspended in ice-cold aq trifluoroacetic acid
(80%, v/v, 550 mL) and then stirred at 0 °C for 4 h, whereupon the
mixture was evaporated to dryness. The crude residue was
coevaporated with 1,2-DCE (2 × 100 mL) affording crude hemiacetal
3 which was used directly in the next step. TLC (50% EtOAc in PE, v/
v): Rf (2) = 0.5, Rf (3) = 0.3. Crude diol 3 (117 g) was dissolved in
anhydrous pyridine (500 mL) and then cooled to 0 °C. After dropwise
addition of Ac2O (100 mL, 1.10 mol) over 30 min, stirring was
continued for 40 h during which the reaction was allowed to heat to rt.
Ice (150 mL) and EtOAc (400 mL) were added. The organic phase
was evaporated to dryness, and then coevaporated with toluene (2 ×
100 mL). The resulting residue was taken up in EtOAc (400 mL) and
the resulting mixture washed with satd aq NaHCO3 (2 × 500 mL).
The combined aq phase was back-extracted with EtOAc (2 × 200
mL). The combined org. phase was dried over MgSO4 and then
evaporated to dryness. After workup, analytical TLC showed some
remaining diol 3. The residue was dissolved in anhydrous pyridine
(500 mL) and then cooled to 0 °C. Ac2O (25 mL, 274 mmol) was
dropwise added over 10 min and the reaction mixture stirred for 18 h.
The reaction mixture was cooled to 0 °C and then partitioned between
ice (150 mL) and EtOAc (200 mL). The separated org phase was
concentrated, then coevaporated with toluene (3 × 200 mL). The
resulting residue was partitioned between EtOAc (400 mL) and satd
aq NaHCO3 (300 mL). The aq phases of the two reactions (i.e., from
reacting a total of 334 g of crude diol 3) were combined and extracted
with EtOAc (2 × 200 mL). The org. phases were combined,
evaporated to dryness and then coevaporated with MeCN (2 × 150
mL) to afford diacetate 4 (274.82 g, two anomers, 98% from 1) as a
brown syrup which was used directly in the next step. TLC (3%
MeOH in DCM, v/v): Rf (3) = 0.1, Rf (4) = 0.6 and 0.7. Analytical
data were identical to those previously reported.50
1-(2-O-Acetyl-3-O-benzyl-4-C-methanesulfonoxymethyl-5-O-
methanesulfonyl-β-D-erythro-pentofuranosyl)thymine (5).50 Five
reaction batches (94−118 mmol) were used for the steps 4→8, a
representative protocol is given here: Coupling sugar 4 (57.28 g, 112
mmol) and thymine (28.30 g, 224 mmol) were coevaporated with
MeCN (2 × 100 mL) and then suspended in anhydrous MeCN (450
mL). N,O-bis(Trimethylsilyl)acetamide (97 mL, 393 mmol) was
added at rt, and the stirred reaction mixture was heated under reflux
for 20 min during which time the reaction mixture became
homogeneous. The reaction mixture was cooled to 0 °C and TMSOTf
(51 mL, 280 mmol) was added dropwise over 20 min whereupon the
resulting mixture was stirred at 60 °C for 92 h. After cooling to 0 °C,
the reaction mixture was poured into satd aq NaHCO3 (250 mL) and
then concentrated to approx 250 mL which led to precipitation. To
the resulting suspension was added DCM (400 mL) and brine (200
mL). The mixture was filtered and the precipitate washed with DCM
(2 × 75 mL). The combined org. phase was washed with satd aq
NaHCO3 (250 mL) and the combined aq phase back-extracted with
DCM (2 × 75 mL). The combined org. phase was evaporated to
dryness and the residue subsequently coevaporated with MeCN (100
CONCLUSION
■
A multigram scale synthesis of thymine intermediate 17 has
been realized from starting sugar 1 in fifteen steps in an overall
yield of 73%, with only five purification steps necessary. One
step conversion to key thymine intermediate 18 was achieved
in 95% yield, whereas the two step conversion to 5-
methylcytosine key intermediate 20 proceeded in 58% yield.
With the procedures disclosed herein, highly efficient larger
scale routes have been established allowing convenient and
high-yielding syntheses of N2′-functionalized thymine and 5-
methylcytosine amino-LNA phosphoramidites in only two and
three divergent steps, respectively.
EXPERIMENTAL SECTION
■
General Experimental Section. All reagents and solvents were of
analytical grade and used without further purification as obtained from
commercial suppliers, except for DCM and POCl3 which were distilled
before use. Petroleum ether of distillation range 60−80 °C was used.
Acetonitrile for use in anhydrous reactions was dried through storage
over activated 3 Å molecular sieves. DCM, 1,2-DCE, 1,4-dioxane,
DMF, DBU, triethylamine, and pyridine for use in anhydrous reactions
were dried through storage over activated 4 Å molecular sieves.
Reactions were conducted under an atmosphere of argon or nitrogen
whenever anhydrous solvents were used. All reactions were monitored
by thin-layer chromatography (TLC) using silica gel coated plates
(analytical SiO2-60, F-254). The TLC plates were visualized under UV
light and by dipping in either a 5% conc. solution of sulfuric acid in
abs. ethanol (v/v) or a solution of molybdato-phosphoric acid (12.5 g/
L) and cerium(IV)sulfate (5 g/L) in 3% conc. sulfuric acid in water
(v/v), followed by heating with a heat gun. Silica gel column
chromatography was performed using an automated purification
system or manually with silica gel 60 (particle size 0.040−0.063 mm,
Merck) using moderate pressure (pressure ball). After column
chromatography, appropriate fractions were pooled, evaporated
under reduced pressure and dried at high vacuum for at least 12 h
to give products in >95% purity unless otherwise stated. Evaporation
of solvents was carried out under reduced pressure at a temperature
1
below 40 °C. H NMR, 13C NMR, 19F NMR and 31P NMR were
recorded at 400 MHz, 101 MHz, 376 MHz, and 162 MHz
respectively. Chemical shifts are reported in ppm relative to deuterated
solvent as internal standard (δH: DMSO-d6 2.50 ppm; δC: DMSO-d6
39.52 ppm) or external standard (δF and δP). Coupling constants are
reported in Hz. Exchangeable protons were detected by disappearance
of peaks upon addition of D2O. Assignments of NMR spectra are
based on correlation spectroscopy (COSY, HSQC, and HMBC
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i
mL) to afford crude nucleoside 5 (64.18 g) as a brown foam, which
was used directly in the next step. TLC (5% MeOH in DCM, v/v): Rf
(4) = 0.7, Rf (5) = 0.4. Analytical data were identical to those
previously reported.49
1-(3-O-Benzyl-5-O-methanesulfonyl-4-C-methanesulfonyloxy-
methyl-β-D-erythro-pentofuranosyl)thymine (6).43 Crude acetate 5
(64.18 g) was dissolved in satd methanolic ammonia (560 mL) at 0 °C
and then stirred 5 h. The reaction mixture was evaporated to dryness
whereupon EtOAc (400 mL) and brine (200 mL) were added. The
aqueous phase was back-extracted with EtOAc (2 × 100 mL) and the
combined organic phase evaporated to dryness affording crude alcohol
6 (63.57 g) as a brown foam, which was used directly in the next step.
TLC (10% MeOH in DCM, v/v): Rf (5) = 0.6, Rf (6) = 0.5. Analytical
data were identical to those previously reported.43
foam. TLC (10% PrOH in CHCl3, v/v): Rf (8) = 0.3, Rf (9) = 0.5.
Analytical data were identical to those previously reported.43
1-(3-O-Benzyl-5-O-methanesulfonyl-4-C-methanesulfonyloxy-
methyl-2-O-trifluoromethanesulfonyl-β-D-threo-pentofuranosyl)-
thymine (10).43 Alcohol 9 (44.82 g, 83.8 mmol) was coevaporated
with 1,2-DCE (100 mL) and then dissolved in anhydrous DCM (1250
mL). DMAP (20.47 g, 168 mmol) and anhydrous pyridine (70 mL,
865 mmol) were added and the resulting solution cooled to −70 °C
with stirring. Triflic anhydride (19.9 mL, 117 mmol) was added
dropwise over 20 min, and the reaction mixture was stirred at −70 °C
for additional 10 min and then heated to 0 °C. After stirring for 4 h at
0 °C, aq HCl (2 M, 500 mL, 1.0 mol) was added, and the org phase
extracted with DCM (100 mL). The combined org phase was washed
with aq HCl (2 M, 500 mL, 1.0 mol) and the aq phase back-extracted
with DCM (100 mL). The combined organic phase was washed with
satd aq NaHCO3 (200 mL) and the aq phase back-extracted with
DCM (100 mL). The combined organic phase was dried over MgSO4
and then adsorbed directly onto silica gel and purified by DCVC
(EtOAc) to give the desired triflate 10 (53.96 g, 97%) as a white foam.
1-(3-O-Benzyl-2,5-di-O-methanesulfonyl-4-C-methanesulfony-
loxymethyl-β-D-erythro-pentofuranosyl)thymine (7).51 Crude alco-
hol 6 (63.57 g) was dissolved in a mixture of anhydrous DCM (300
mL) and anhydrous pyridine (300 mL). After cooling to 0 °C, MsCl
(11.0 mL, 143 mmol) was added dropwise over 5 min with stirring
whereupon the reaction mixture was heated to rt. After stirring at rt for
5 h the reaction mixture was poured into satd aq NaHCO3 (200 mL)
and concentrated to approximately 200 mL. DCM (400 mL), brine
(200 mL) and water (100 mL) were added, the org phase separated
and the aq phase back-extracted with DCM (2 × 100 mL). The
combined organic phase was evaporated to dryness and the residue
coevaporated with toluene (2 × 100 mL) to give a mixture of desired
mesylate 7 and 2,2′-anhydro derivative 8 (76.88 g) as a brown foam,
which was used directly in the next step. TLC (10% MeOH in DCM,
v/v): Rf (6) = 0.6, Rf (7) = 0.5, Rf (8) = 0.4. Analytical data were
identical to those previously reported.51
2,2′-Anhydro-1-(3-O-benzyl-5-O-methanesulfonyl-4-C-methane-
sulfonyloxymethyl-β-D-threo-pentofuranosyl)thymine (8).43 The
crude mixture of mesylate 7 and 2,2′-anhydro derivative 8 (76.88 g)
was coevaporated with acetonitrile/toluene (2 × 150 mL, 67:33 v/v)
and then dissolved in anhydrous MeCN (550 mL). The resulting
mixture was cooled to 0 °C under mechanical stirring, DBU (20.6 mL,
138 mmol) was added and the reaction mixture was stirred for 24 h
during which time the reaction was allowed to heat to rt. Extensive
precipitation was observed and further facilitated by subsequently
concentrating the reaction mixture to approximately 250 mL and
cooling to −20 °C The precipitate was filtered off and washed with
ice-cold MeCN (2 × 25 mL). The precipitate was dried under vacuum
affording the desired anhydro derivative 8 (33.37 g, 57%) as a white
solid. The mother liquors from the five reaction batches were
combined and then concentrated leading to extensive precipitation.
The precipitate was filtered off to afford the desired anhydro derivative
8 (92.64 g) as a white solid. The combined mother liquor was further
concentrated affording a residue which was purified by silica gel
column chromatography (0−10% iPrOH in CHCl3, v/v) affording the
desired anhydro derivative 8 (5.65 g) as a white foam. In total, 242.89
g (470 mmol, 86% from 1) of the desired anhydro derivative 8 was
isolated. TLC (10% MeOH in DCM, v/v): Rf (7) = 0.5, Rf (8) = 0.4.
Analytical data were identical to those previously reported.43
1-(3-O-Benzyl-5-O-methanesulfonyl-4-C-methanesulfonyloxy-
methyl-β-D-threo-pentofuranosyl)thymine (9).43 2,2′-Anhydro de-
rivative 8 (44.01 g, 85.2 mmol) was suspended in a stirred mixture of
acetone (250 mL) and aq H2SO4 (1.0 M, 220 mL, 220 mmol). The
resulting mixture was heated under reflux for 3 h resulting in a
transparent yellow solution. Subsequent cooling to 0 °C induced
extensive precipitation. The reaction mixture was then neutralized by
careful addition of sat, aq, NaHCO3 (250 mL) under stirring. The
suspension was filtered and the mother liquor concentrated to
approximately 500 mL, leading to further precipitation. The combined
precipitate was washed with water (3 × 400 mL). The combined aq
phase was extracted with DCM (100 mL) and the precipitate and org.
phase evaporated to dryness and coevaporated with 1,2-DCE (4 × 200
mL). The resulting solid was adsorbed on silica gel and purified by
DCVC (dry column vacuum chromatography) (0−6% MeOH in
DCM, v/v), affording the desired alcohol 9 (44.82 g, 98%) as a white
i
TLC (10% PrOH in CHCl3, v/v): Rf (9) = 0.5, Rf (10) = 0.6.
Analytical data were identical to those previously reported.43
1-(2-Azido-3-O-benzyl-2-deoxy-2,5-di-O-methanesulfonyl-4-C-
methanesulfonyloxymethyl-β-D-erythro-pentofuranosyl)thymine
(11).43 Triflate 10 (53.96 g, 80.9 mmol) was dissolved in anhydrous
DMF (400 mL) and NaN3 (7.91 g, 122 mmol) was added at 0 °C. The
reaction mixture was stirred at rt for 16 h and then slowly added to
10% brine (v/v, 3000 mL) which induced extensive precipitation. The
precipitate was filtered off and washed with water (2 × 400 mL). The
combined aqueous phase was extracted with EtOAc (2 × 500 mL).
The precipitate was taken up in the organic phase which was washed
with 10% brine (v/v, 2 × 300 mL), dried over MgSO4 and evaporated
to dryness to afford crude azide 11 (46.22 g, DMF as impurity) as a
white foam. TLC (70% EtOAc in PE, v/v): Rf (10) = 0.3, Rf (11) =
0.25. Analytical data were identical to those previously reported.43
1-(5-O-Methanesulfonyl-4-C-methanesulfonyloxymethyl-3-O-
benzyl-2-deoxy-2-azido-β-D-erythro-pentofuranosyl)-4-azido-5-
methylpyrimidin-2(1H)-one (12). Alcohol 9 (5.19 g, 9.71 mmol) was
coevaporated with pyridine (2 × 25 mL), then suspended in anh DCM
(50 mL) and anh pyridine (16 mL, 198 mmol) and DMAP (4.73 g,
38.7 mmol) was added under stirring at 0 °C, resulting in a clear
solution, followed by addition of triflic anhydride (3.3 mL, 19.4
mmol). After stirring for 2 h at 0 °C the reaction mixture was poured
into sat aq NaHCO3 (50 mL) and the organic phase washed
consecutively with aq HCl (1.0 M, 2 × 50 mL) and sat aq NaHCO3
(50 mL), then dried over MgSO4, and evaporated to dryness to afford
a residue which was dried over high vacuum for 48 h, then dissolved in
anh DMF (50 mL) under argon. 15-crown-5 (1.93 mL, 9.72 mmol)
and NaN3 (694 mg, 10.7 mmol) was added, then the reaction mixture
heated to 80 °C. After stirring for 19 h, the reaction mixture was
poured into water (100 mL) and EtOAc (100 mL) was added. The
organic phase was washed with sat. aq. NaHCO3 (2 × 100 mL), then
dried over MgSO4, and evaporated to dryness. The resulting residue
was purified by column chromatography (0−7% MeOH in DCM) to
afford azide 11 (2.59 g, 48%) and diazide 12 (1.19 g, 21%) as white
foams. Data for diazide 12: TLC (70% EtOAc in PE): Rf (10) = 0.3, Rf
(11) = 0.25, Rf (12) = 0.3; TLC (10% MeOH in DCM): Rf (10) =
0.3, Rf (11) = 0.3, Rf (12) = 0.5. 1H NMR (DMSO-d6) δ 7.64 (d, J =
1.2, 1H, H6), 7.31−7.47 (m, 5H, H2Bn, H3Bn, H4Bn, H5Bn, H6Bn), 6.20
(d, J = 4.9, 1H, H1′), 4.83 (dd, J = 4.9, 6.5, 1H, H2′), 4.77 (s, 2H,
CH2Ph), 4.69 (d, J = 6.5, 1H, H3′), 4.58 (d, J = 11.4, 1H, H5′A/H5″A),
4.57 (d, J = 10.9, 1H, H5′A/H5″A), 4.51 (d, J = 10.9, 1H, H5′B/H5″B),
4.39 (d, J = 11.4, 1H, H5′B/H5″B), 3.33 (s, 3H, SCH3), 3.23 (s, 3H,
SCH3), 2.31 (d, J = 1.2, 3H, 5-CH3). 13C NMR (DMSO-d6) δ 151.8
(C4), 142.6 (C2), 137.0 (C1Bn), 131.4 (C6), 128.4 (C2Bn, C6Bn),
128.1 (C4Bn), 128.0 (C3Bn, C5Bn), 103.4 (C5), 88.5 (C1′), 84.2 (C4′),
78.2 (C3′), 73.9 (CH2Ph), 68.0 (C5′/C5″), 67.7 (C5′/C5″), 63.5
(C2′), 37.0 (SCH3), 36.9 (SCH3), 12.5 (5-CH3). ESI-HRMS m/z
585.1187 ([M + H]+, C20H25N8O9S2 Calcd 585.1180).
+
(1R,3R,4R,7S)-7-Benzyloxy-1-methanesulfonyloxymethyl-3-(thy-
min-1-yl)-2-oxa-5-azabicyclo[2.2.1]heptane (13).43 Crude azide 11
10723
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Article
(46.22 g) was dissolved in THF (400 mL), and then aq NaOH (2M,
100 mL, 200 mmol) was added. The resulting mixture was cooled to 0
°C with stirring under an atmosphere of argon. PMe3 in THF (1M,
100 mL, 100 mmol) was added slowly leading to distinct effervescence.
The resulting mixture was allowed to heat to rt and stirred for 14 h.
The reaction mixture was concentrated to approximately 100 mL and
then 33% brine (v/v, 300 mL) and 20% MeOH in DCM (v/v, 800
mL) were added. The resulting mixture was neutralized by addition of
aq HCl (2 M, 75 mL) and satd aq NaHCO3 (10 mL). The phases
were separated and the aq phase extracted with 20% MeOH in DCM
(v/v, 2 × 200 mL). The combined org phase was evaporated to
dryness affording crude derivative 13 (35.26 g, POMe3 as impurity) as
a slightly yellow solid which was used in the next step without
purification. TLC (10% MeOH in EtOAc, v/v): Rf (11) = 0.7, Rf
(intermediate) = 0.4, Rf (13) = 0.3. Analytical data were identical to
those previously reported.43
(1R,3R,4R,7S)-1-Benzoyloxymethyl-7-benzyloxy-3-(thymin-1-yl)-
2-oxa-5-azabicyclo[2.2.1]heptane (14).43 A three-neck RB equipped
with reflux condenser and mechanical stirring was charged with 18-
crown-6 (25.47 g, 97 mmol). Crude mesylate 13 (35.26 g) was
suspended in dioxane (800 mL) and added to the crown ether with
stirring. KOBz (25.82 g, 161 mmol) was added and the reaction
mixture heated at 80 °C for 18 h. The reaction mixture was cooled to
rt, evaporated to dryness and to the resulting residue was added
EtOAc (500 mL) and water (500 mL). The aq phase was extracted
with EtOAc (2 × 100 mL) and the combined organic phase dried over
MgSO4 and evaporated to dryness. The resulting residue was purified
by DCVC (0−6% MeOH in DCM, v/v) to afford the desired benzoate
ester 14 (35.35 g, 94% from 10) as a white foam. TLC (10% iPrOH in
CHCl3, v/v): Rf (13) = 0.2, Rf (14) = 0.3. Analytical data were
identical to those previously reported.43
H5″B), 3.59 (d, J = 11.6, 1HI, H5″A), 3.42 (d, J = 11.6, 1HI, H5″B),
1.79 (d, J = 0.9, 3H, 5-CH3). 13C NMR (DMSO-d6) δ 164.0 (C4I),
163.9 (C4II), 155.0 (q, J = 36.4, COCF3,II), 154.9 (q, J = 36.8,
COCF3,I), 149.86 (C2II), 149.85 (C2I), 137.44 (C1Bn,II), 137.42
(C1Bn,I), 134.7 (C6II), 134.6 (C6I), 128.30 (C3Bn,I, C5Bn,I), 128.27
(C3Bn,II, C5Bn,II), 127.7 (C4Bn), 127.3 (C2Bn,II, C6Bn,II), 127.2 (C2Bn,I
,
C6Bn,I), 115.9 (q, J = 288.5, CF3,II), 115.8 (q, J = 287.8, CF3,I), 108.4
(C5II), 108.3 (C5I), 88.5 (C4′II), 87.1 (C4′I), 85.6 (C1′I), 85.1 (C1′II),
75.7 (C3′I), 74.2 (C3′II), 71.44 (CH2PhII), 71.43 (CH2PhI), 61.9
(C2′I), 60.3 (C2′II), 56.4 (C5′), 52.6 (C5″I), 52.4 (C5″II), 12.50 (5-
CH3,I), 12.47 (5-CH3,II). 19F NMR (DMSO-d6) δ −70.1 (CF3,I),
+
−71.6 (CF3,II). ESI-HRMS m/z 456.1386 ([M + H]+, C20H21F3N3O6
Calcd 456.1377).
(1R,3R,4R,7S)-7-Benzyloxy-1-(4,4′-dimethoxytrityl)oxymethyl-3-
(thymin-1-yl)-5-trifluoroacetyl-2-oxa-5-azabicyclo[2.2.1]heptane
(17). Crude alcohol 16 was dissolved in a mixture of anhydrous
pyridine (180 mL) and anhydrous DCM (180 mL). DMTCl (29.03 g,
85.7 mmol) was added and the resulting mixture was stirred for 18 h at
rt. MeOH (5 mL) was added and the reaction mixture evaporated to
dryness. The resulting residue was coevaporated with toluene (2 × 50
mL) whereupon DCM (300 mL) was added. After washing with satd
aq NaHCO3 (150 mL) and brine (150 mL), the combined aqueous
phase was extracted with DCM (2 × 50 mL). The combined organic
phase was evaporated to dryness and the resulting residue purified by
DCVC (0−4% MeOH in DCM, v/v) to afford a rotameric mixture of
the desired nucleoside 17 (51.64 g, 95% from 15, 2:3 ratio between
rotamers according to 1H NMR) as a yellow foam. TLC (5% iPrOH in
CHCl3, v/v): Rf (16) = 0.1, Rf (17) = 0.5. Found: C, 64.6; H, 5.1; N,
5.6. Calcd for C41H38F3N3O8: C, 65.0; H, 5.1; N, 5.5. 1H NMR
(DMSO-d6) δ 11.46 (s, 1HII, ex, N3−H), 11.44 (s, 1HI, ex, N3−H),
7.56 (d, J = 1.2, 1HII, C6), 7.55 (d, J = 1.2, 1HI, C6), 7.34−7.38 (m,
2H, H2″DMT, H6″DMT), 7.28−7.33 (m, 5H, H3″DMT, H5″DMT, H3Bn,
(1R,3R,4R,7S)-7-Benzyloxy-1-hydroxymethyl-3-(thymin-1-yl)-2-
oxa-5-azabicyclo[2.2.1]heptane (15).43 Benzoate ester 14 (35.35 g,
76.3 mmol) was dissolved in freshly prepared methanolic ammonia
(760 mL) at 0 °C and stirred at 6 °C for 4 days. Silica gel (∼100 mL)
was added and the reaction mixture was then evaporated to dryness.
The residue was purified by DCVC (0−10% MeOH in DCM, v/v) to
afford the desired alcohol 15 (25.64g, 94%) as a white foam. TLC
(10% MeOH in EtOAc, v/v): Rf (14) = 0.3, Rf (15) = 0.2. Analytical
data were identical to those previously reported.43
H4Bn, H5Bn), 7.22−7.27 (m, 5H, H2DMT, H6DMT, H2′DMT, H6′DMT
,
,
H4″DMT), 7.18−7.22 (m, 2H, H2Bn, H6Bn), 6.84−6.91 (m 4H, H3DMT
H5DMT, H3′DMT, H5′DMT), 5.700 (s, 1HI, H1′), 5.679 (s, 1HII, H1′),
5.22 (s, 1HII, H2′), 5.06 (s, 1HI, H2′), 4.63 (d, J = 11.9, 1HI, CH2PhA),
4.62 (d, J = 11.9, 1H, 1HII, CH2PhA), 4.57 (d, J = 11.9, 1H, CH2PhB),
4.45 (s, 1H, H3′), 3.733 (s, 3HI, OCH3), 3.729 (s, 3HII, OCH3), 3.723
(s, 3HI, OCH3), 3.720 (s, 3HII, OCH3), 3.61−3.70 (m, 1H, H5″A),
3.41−3.52 (m, 3H, H5′A+B, H5″B), 1.47−1.50 (m, 3H, 5-CH3). 13C
(1R,3R,4R,7S)-7-Benzyloxy-1-hydroxymethyl-3-(thymin-1-yl)-5-
trifluoroacetyl-2-oxa-5-azabicyclo[2.2.1]heptane (16). Amino alco-
hol 15 (25.68 g, 71.5 mmol) was coevaporated with pyridine (50 mL)
and then dissolved in a mixture of anhydrous pyridine (180 mL) and
anhydrous DCM (180 mL). After cooling to −70 °C with stirring,
trifluoroacetic anhydride (22.5 mL, 159 mmol) was added dropwise
over 30 min and the resulting mixture was stirred at −70 °C for 10
min, whereafter the reaction mixture was heated to 0 °C and further
stirred for 30 min. The reaction mixture was evaporated to dryness and
the residue coevaporated with toluene (2 × 50 mL). To the resulting
residue was added DCM (300 mL) and satd aq NaHCO3 (175 mL),
and the resulting biphasic mixture was then stirred at rt for 10 min
which resulted in distinct effervescence. The two phases were
separated and the org phase washed with satd aq NaHCO3 (175
mL). The resulting biphasic mixture was stirred for 10 min and then
separated. The combined aq phase was extracted with DCM (2 × 50
mL), and the combined organic phase then evaporated to dryness. The
residue was coevaporated with pyridine to afford a rotameric mixture
NMR (DMSO-d6) δ 163.94 (C4I), 163.86 (C4II), 158.24 (C4DMT,I,
C4′DMT,I), 158.20 (C4DMT,II, C4′DMT,II), 155.0 (q, J = 36.6, COCF3,II),
154.8 (q, J = 37.1, COCF3,I), 149.81 (C2II), 149.80 (C2I), 144.4
(C1″DMT), 137.1 (C1Bn,II), 137.0 (C1Bn,I), 134.88 (C1DMT,I/C1′DMT,I),
134.86 (C1DMT,II/C1′DMT,II), 134.77 (C1DMT,II/C1′DMT,II), 134.76
(C1DMT,I/C1′DMT,I), 133.92 (C6II), 133.86 (C6I), 129.7 (C2DMT
,
,
C6DMT, C2′DMT, C6′DMT), 128.33 (C3Bn,I, C5Bn,I), 128.29 (C3Bn,II
C5Bn,II), 128.0 (C3″DMT, C5″DMT), 127.9 (C4Bn,I), 127.8 (C4Bn,II),
127.53 (C2Bn,II, C6Bn,II, C2″DMT,II, C6″DMT,II), 127.51 (C2Bn,I, C6Bn,I
C2″DMT,I, C6″DMT,I), 126.9 (C4″DMT), 115.80 (q, J = 288.7, CF3,II),
115.76 (q, J = 287.3, CF3,I), 113.38 (C3DMT,I, C5DMT,I/C3′DMT,I
C5′DMT,I), 113.36 (C3DMT,II, C5DMT,II/C3′DMT,II, C5′DMT,II), 113.34
(C3DMT,I, C5DMT,I/C3′DMT,I, C5′DMT,I), 113.33 (C3DMT,II, C5DMT,II
,
,
/
C3′DMT,II, C5′DMT,II), 108.7 (C5II), 108.6 (C5I), 87.1 (C4′II), 86.00
(CAr3,II), 85.97 (CAr3,I), 85.9 (C1′I), 85.6 (C4′I), 85.4 (C1′II), 75.8
(C3′I), 74.3 (C3′II), 71.3 (CH2PhII), 71.2 (CH2PhI), 61.7 (C2′I), 60.1
(C2′II), 58.5 (C5′I), 58.4 (C5′II), 55.0 (OCH3), 52.9 (C5″I), 52.7
(C5″II), 12.47 (5-CH3,I), 12.45 (5-CH3,II). 19F NMR (DMSO-d6) δ
−70.1 (CF3,I), −71.6 (CF3,II). ESI-HRMS m/z 780.2507 ([M + Na]+,
C41H38F3N3O8Na+ Calcd 780.2503).
(1R,3R,4R,7S)-7-Hydroxy-1-(4,4′-dimethoxytrityl)oxymethyl-3-
(thymin-1-yl)-2-oxa-5-azabicyclo[2.2.1]heptane (18). Nucleoside 17
(20.04 g, 26.4 mmol) was dissolved in MeOH (500 mL) and the
resulting mixture was evacuated and an atmosphere of nitrogen was
applied. Evacuation and nitrogen atmosphere application was repeated
three times. 20% Pd(OH)2/C (1.39 g) and ammonium formate (11.63
g, 184 mmol) were added and the reaction heated under reflux with
stirring for 3 h. The reaction mixture was cooled to rt and filtered
through a short Celite pad. The Celite pad was washed thoroughly
with MeOH (3 × 50 mL) and DCM (3 × 50 mL) and the resulting
1
of crude alcohol 16 (40:60 ratio according to H NMR) as a brown
foam which was used without further purification in the next step.
TLC (10% MeOH in DCM, v/v): Rf (15) = 0.2, Rf (16) = 0.5. Found:
C, 52.4; H, 4.4; N, 9.1. Calcd for C20H20N3O6F3: C, 52.7; H, 4.4; N,
9.1. 1H NMR (DMSO-d6) δ 11.39 (s, 1HII, ex, N3−H), 11.37 (s, 1HI,
N3−H), 7.68 (d, J = 0.9, 1HI, H6), 7.66 (d, J = 0.9, 1HII, H6), 7.26−
7.35 (m, 3H, H3Bn, H4Bn, H5Bn), 7.21−7.25 (m, 2H, H2Bn, H6Bn), 5.66
(s, 1H, H1′), 5.45−5.52 (m, 1H, ex, 5′−OH), 5.09 (s, 1HII, H2′), 4.92
(s, 1HI, H2′), 4.63 (d, J = 11.8, 1HI, CH2PhA), 4.62 (d, J = 11.7, 1H,
1HII, CH2PhA), 4.56 (d, J = 11.8, 1HI, CH2PhB), 4.53 (d, J = 11.7, 1H,
1HII, CH2PhB), 4.18 (s, 1HI, H3′), 4.17 (s, 1HII, H3′), 3.77−3.91 (m,
2H, H5′A+B), 3.75 (d, J = 10.0, 1HII, H5″A), 3.67 (d, J = 10.0, 1HII,
10724
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combined mixture was evaporated to dryness. The resulting residue
was suspended in MeOH (100 mL) and filtered through a glass filter.
The white precipitate was washed with cold MeOH (2 × 100 mL) and
dried under high vacuum to afford the desired amino alcohol 18
(10.37 g) as a white solid. The methanolic phase was evaporated to
dryness affording a white solid, which was purified by DCVC (0−12%
MeOH in DCM, v/v) to afford the desired amino alcohol 18 (4.21 g)
as a white solid (total yield of amino alcohol 18: 14.58 g, 96%). TLC
C6Bn), 126.9 (C4″DMT), 115.85 (q, J = 288.7, CF3,II), 115.78 (q, J =
287.9, CF3,I), 113.39 (C3DMT,I, C5DMT,I/C3′DMT,I, C5′DMT,I), 113.37
(C3DMT,I, C5DMT,I/C3′DMT,I, C5′DMT,I), 113.35 (C3DMT,II, C5DMT,II
,
C3′DMT,II, C5′DMT,II), 101.10 (C5II), 101.06 (C5I), 86.8 (C4′II), 86.4
(C1′I), 86.01 (C1′II), 85.99 (CAr3,II), 85.96 (CAr3,I), 85.4 (C4′I), 75.6
(C3′I), 74.1 (C3′II), 71.35 (CH2PhII), 71.28 (CH2PhI), 61.6 (C2′I),
60.0 (C2′II), 58.54 (C5′I), 58.45 (C5′II), 55.0 (OCH3), 52.9 (C5″I),
52.7 (C5″II), 13.59 (5-CH3,I), 13.57 (5-CH3,II). 19F NMR (DMSO-d6)
δ −69.9 (CF3,I), −71.6 (CF3,II) ESI-HRMS m/z 757.2872 ([M + H]+,
1
(10% MeOH in DCM, v/v): Rf (17) = 0.5, Rf (18) = 0.3. H NMR
+
(DMSO-d6) δ 11.35 (s, 1H, ex, N3−H), 7.62 (d, J = 0.9, 1H, H-6),
C41H40F3N4O7 Calcd 757.2844).
7.42−7.45 (m, 2H, H2″DMT, H6″DMT), 7.28−7.35 (m, 6H, H2DMT
,
(1R,3R,4R,7S)-7-Hydroxy-1-(4,4′-dimethoxytrityl)oxymethyl-3-(5-
methylcytosin-1-yl)-2-oxa-5-azabicyclo[2.2.1]heptane (20). Thy-
mine nucleoside 17 (19.05 g, 25.1 mmol) was converted to crude 5-
methylcytosine nucleoside 19 using the conditions described above,
but proceeding with the following step without purification. Crude
nucleoside 19 was dissolved in MeOH (500 mL) followed by
evacuation and introduction of an N2 atmosphere. Evacuation and
nitrogen atmosphere application was repeated three times. 20%
Pd(OH)2/C (1.46 g) and ammonium formate (11.34 g, 180 mmol)
were added with stirring and the reaction mixture was heated under
reflux for 2.5 h. Then the reaction mixture was cooled to rt and filtered
through a short Celite pad. The Celite pad was washed thoroughly
with MeOH (3 × 50 mL) and DCM (3 × 50 mL) and the resulting
mixture was evaporated to dryness. The resulting residue was purified
by silica gel column chromatography (10−18% MeOH in DCM, v/v)
affording a light yellow solid (9.88 g). This solid was dissolved in 2%
MeOH in CHCl3 (v/v, 500 mL) and the org. phase was washed with
aq K2CO3 (0.1 M, 1.0 L). The aq phase was back-extracted with
CHCl3 (6 × 100 mL) and 10% MeOH in CHCl3 (v/v, 2 × 100 mL).
The combined org phase was evaporated to dryness. The resulting
residue was purified by silica gel column chromatography (10−20%
MeOH in DCM, v/v) affording the desired nucleoside 20 (8.37 g,
58%), as a white foam. TLC (20% MeOH in DCM, v/v): Rf (19) =
0.8, Rf (20) = 0.1. 1H NMR (DMSO-d6) δ 7.59 (s, 1H, H6), 7.42−47
H6DMT, H2′DMT, H6′DMT, H3″DMT, H5″DMT), 7.22−7.27 (m, 1H,
H4″DMT), 6.89−6.92 (m, 4H, H3DMT, H5DMT, H3′DMT, H5′DMT), 5.40
(d, J = 4.5, 1H, ex, 3′−OH), 5.35 (s, 1H, H1′), 4.06 (d, J = 4.5, 1H,
H3′), 3.74 (s, 6H, OCH3), 3.35 (d, J = 10.8, 1H, H5′A), 3.31 (s, 1H,
H2′), 3.28 (d, J = 10.8, H5′B), 2.84 (d, J = 9.9, 1H, H5″A), 2.63 (d, J =
9.9, 1H, H5″B), 1.50 (d, J = 0.9, 3H, 5-CH3). 13C NMR (DMSO-d6) δ
163.8 (C4), 158.2 (C4DMT, C4′DMT), 149.9 (C2), 144.8 (C1″DMT),
135.5 (C1DMT/C1′DMT), 135.2 (C1DMT/C1′DMT), 134.7 (C6), 129.8
(C2DMT, C6DMT/C2′DMT, C6′DMT), 129.7 (C2DMT, C6DMT/C2′DMT
,
C6′DMT), 127.9 (C3″DMT, C5″DMT), 127.7 (C2″DMT, C6″DMT), 126.8
(C4″DMT), 113.26 (C3DMT, C5DMT/C3′DMT, C5′DMT), 113.25 (C3DMT
,
C5DMT/C3′DMT, C5′DMT), 108.1 (C5), 88.1 (C1′), 87.9 (C4′), 85.6
(CAr3), 69.5 (C3′), 61.9 (C2′), 59.7 (C5′), 55.0 (OCH3), 49.9 (C5″),
+
12.3 (5-CH3). ESI-HRMS m/z 572.2375 ([M + H]+, C32H34N3O7
Calcd 572.2391).
(1R,3R,4R,7S)-7-Benzyloxy-1-(4,4′-dimethoxytrityl)oxymethyl-3-
(5-methylcytosin-1-yl)-5-trifluoroacetyl-2-oxa-5-azabicyclo[2.2.1]-
heptane (19). Thymine nucleoside 17 (5.01 g, 6.61 mmol) was
coevaporated with MeCN (20 mL) and then dissolved in anhydrous
MeCN (120 mL). Anhydrous Et3N (16.0 mL, 114 mmol) and 1,2,4-
triazole (5.46 g, 79.1 mmol) were added and the resulting mixture was
cooled to 0 °C with stirring. Freshly distilled phosphorus oxychloride
(1.85 mL, 19.8 mmol) was added dropwise and after stirring for 10
min at 0 °C the reaction mixture was heated to r.t and stirred for
additional 60 min when analytical TLC indicated full conversion to a
fluorescent intermediate tentatively assigned as 1-((1R,3R,4R,7S)-7-
benzyloxy-1-(4,4′-dimethoxytrityl)oxymethyl-5-trifluoroacetyl-2-oxa-5-
azabicyclo[2.2.1]heptan-3-yl)-5-methyl-4-(1H-1,2,4-triazol-1-yl)-
pyrimidin-2(1H)-one [TLC (50% EtOAc in PE, v/v): Rf (17) = 0.3, Rf
(intermediate) = 0.1]. The reaction mixture was partitioned between
half satd aq NaHCO3 (200 mL) and EtOAc (200 mL). The aq phase
was extracted with EtOAc (50 mL) and the combined org phase
washed with brine (50 mL) and dried over MgSO4 followed by
evaporation to dryness. The resulting residue was dissolved in THF
(60 mL) and satd aq NH3 (2.8 mL) was added. After stirring for 60
min, the reaction mixture was evaporated to dryness and the resulting
residue purified by DCVC (0−7% MeOH in DCM, v/v) to afford a
rotameric mixture of 5-methylcytosine nucleoside 19 (5.01 g,
(m, 2H, H2″DMT, H6″DMT), 7.37−7.22 (m, 7H, H2DMT, H6DMT
,
H2′DMT, H6′DMT, H3″DMT, H4″DMT, H5″DMT), 6.95−6.88 (m, 4H,
H3DMT, H5DMT, H3′DMT, H5′DMT), 6.75 (br s, 1H, NH), 5.38 (d, J =
4.1, 1H, 3′−OH), 5.34 (s, 1H, H1′), 4.03 (d, J = 4.1, 1H, H3′), 3.74 (s,
6H, OCH3), 3.35−3.26 (m, 3H, H2′, H5′A+B), 2.83 (d, J = 9.9, 1H,
H5″A), 2.64 (d, J = 9.9, 1H, H5″B), 1.56 (s, 3H, 5-CH3). 13C NMR
(DMSO-d6) δ 165.4 (C4), 158.1 (C4DMT, C4′DMT), 154.8 (C2), 144.8
(C1″DMT), 137.0 (C6), 135.5 (C1DMT/C1′DMT), 135.3 (C1DMT
/
C1′DMT), 129.8 (C2DMT, C6DMT/C2′DMT, C6′DMT), 129.7 (C2DMT
,
,
C6DMT/C2′DMT, C6′DMT), 127.9 (C3″DMT, C5″DMT), 127.8 (C2″DMT
C6″DMT), 126.8 (C4″DMT), 113.3 (C3DMT, C5DMT, C3′DMT, C5′DMT),
100.2 (C5), 88.3 (C1′), 87.5 (C4′), 85.6 (CAr3), 69.3 (C3′), 61.8
(C2′), 59.7 (C5′), 55.0 (OCH3), 50.0 (C5″), 13.4 (5-CH3). ESI-
+
HRMS m/z 571.2562 ([M + H]+, C32H35N4O6 Calcd 571.2551).
(1R,3R,4R,7S)-7-Benzyloxy-1-(4,4′-dimethoxytrityl)oxymethyl-3-
(4-N-benzoyl-5-methylcytosin-1-yl)-5-trifluoroacetyl-2-oxa-5-
azabicyclo[2.2.1]heptane (21a). Nucleoside 19 (4.99 g, 6.59 mmol)
was coevaporated with pyridine (2 × 10 mL) and then dissolved in
anhydrous pyridine (60 mL) at rt. BzCl (1.00 mL, 8.56 mmol) was
added with stirring and the reaction mixture was stirred for 20 h. Then
BzCl (1.00 mL, 8.61 mmol) was added and stirring was continued for
additional 4 h whereupon the reaction mixture was evaporated to
dryness. DCM (200 mL) was added and washed was performed with
satd aq NaHCO3 (100 mL) and brine (100 mL). The org phase was
separated, dried over MgSO4 and evaporated to dryness. The resulting
residue was purified by DCVC (0−50% [30% EtOAc in PE, v/v] in
[50% DCM in PE, v/v], v/v), to afford a rotameric mixture of the
desired protected nucleoside 21a (4.22 g, 74%, 2:3 ratio of rotamers
1
quantitative yield, 2:3 ratio of rotamers according to H NMR) as a
white foam. TLC (5% MeOH in DCM, v/v): Rf (intermediate) = 0.4,
1
Rf (19) = 0.2. H NMR (DMSO-d6) δ 7.56 (s, 1H, H6), 7.36−7.40
(m, 2H, H2″DMT, H6″DMT), 7.22−7.34 (m, 10H, H3Bn, H4Bn, H5Bn,
H2DMT, H6DMT, H2′DMT, H6′DMT, H3″DMT, H4″DMT, H5″DMT), 7.15−
7.19 (m, 2H, H2Bn, H6Bn), 6.85−6.92 (m, 4H, H3DMT, H5DMT
,
H3′DMT, H5′DMT), 5.664 (s, 1HII, H1′), 5.655 (s, 1HI, H1′), 5.20 (s,
1HII, H2′), 5.04 (s, 1HI, H2′), 4.62 (d, J = 11.8, 1HI, CH2PhA), 4.60
(d, J = 11.7, 1HII, CH2PhA), 4.53 (d, J = 11.8, 1H, CH2PhB), 4.42 (s,
1HI, H3′), 4.41 (s, 1HII, H3′), 3.732 (s, 3HI, OCH3), 3.728 (s, 3HII,
OCH3), 3.725 (s, 3HI, OCH3), 3.72 (s, 3HII, OCH3), 3.61−3.70 (m,
1H, H5″A), 3.41−3.53 (m, 3H, H5′A+B, H5″B), 1.56 (s, 3HII, 5-CH3),
1.55 (s, 3HI, 5-CH3). 13C NMR (DMSO-d6) δ 165.7 (C4I), 165.6
(C4II), 158.23 (C4DMT,I, C4′DMT,I), 158.21 (C4DMT,II, C4′DMT,II), 155.1
(q, J = 37.5, COCF3,I), 154.9 (q, J = 36.4, COCF3,II), 154.6 (C2I),
154.5 (C2II), 144.36 (C1″DMT,II), 144.35 (C1″DMT,I), 137.02 (C1Bn,II),
136.95 (C1Bn,I), 136.3 (C6), 135.0 (C1DMT,I/C1′DMT,I), 134.9
1
according to H NMR) as a white foam. TLC (5% MeOH in CHCl3,
v/v): Rf (19) = 0.2, Rf (21a) = 0.8. 1H NMR (DMSO-d6) δ 13.14 (br
s, 1H, ex, 4-NH), 8.18−8.25 (m, 2H, H2Bz, H6Bz), 7.82 (s, 1H, H6),
7.57−7.63 (m, 1H, H4Bz), 7.48−7.54 (m, 2H, H3Bz, H5Bz), 7.36−7.41
(m, 2H, H2″DMT, H6″DMT), 7.24−7.35 (m, 10H, H3Bn, H4Bn, H5Bn,
H2DMT, H6DMT, H2′DMT, H6′DMT, H3″DMT, H4″DMT, H5″DMT), 7.18−
(C1DMT,II/C1′DMT,II), 134.9 (C1DMT,II/C1′DMT,II), 134.8 (C1DMT,I
/
C1′DMT,I), 129.7 (C2DMT, C6DMT, C2′DMT, C6′DMT), 128.32 (C3Bn,I
,
C5Bn,I), 128.29 (C3Bn,II, C5Bn,II), 128.0 (C3″DMT, C5″DMT), 127.9
(C4Bn,I), 127.8 (C4Bn,II), 127.6 (C2″DMT, C6″DMT), 127.5 (C2Bn,
7.23 (m, 2H, H2Bn, H6Bn), 6.85−6.93 (m, 4H, H3DMT, H5DMT,
H3′DMT, H5′DMT), 5.80 (s, 1HI, H1′), 5.79 (m, 1HII, H1′), 5.30 (s,
10725
dx.doi.org/10.1021/jo302036h | J. Org. Chem. 2012, 77, 10718−10728

The Journal of Organic Chemistry
Article
1HII, H2′), 5.15 (s, 1HI, H2′), 4.63 (d, 1HI, J = 12.0, CH2PhA), 4.62
(d, 1HII, J = 12.0, CH2PhA), 4.58 (d, 1H, J = 12.0, CH2PhB), 4.50 (s,
1H, H3′), 3.744 (s, 3HI, OCH3), 3.741 (s, 3HII, OCH3), 3.733 (s, 3HI,
OCH3), 3.729 (s, 3HII, OCH3), 3.64−3.73 (m, 1H, H5″A), 3.46−3.55
(m, 3H, H5′A+B, H5″B), 1.733 (s, 3HI, 5-CH3), 1.725 (s, 3HII, 5-CH3).
61.2 (C2′I), 59.6 (C2′II), 58.5 (C5′I), 58.4 (C5′II), 55.0 (OCH3), 52.9
(C5″I), 52.7 (C5″II), 24.90 (COCH3,I), 24.87 (COCH3,II), 14.2 (5-
CH3). 19F NMR (DMSO-d6) δ −70.0 (CF3,I), −71.6 (CF3,II). ESI-
+
HRMS m/z 799.2947 ([M + H]+, C43H42F3N4O8 Calcd 799.2949).
(1R,3R,4R,7S)-1-(4,4′-Dimethoxytrityloxymethyl)-5-(hexadecano-
yl)-7-hydroxy-3-(4-N-benzoyl-5-methyl-cytosine-1-yl)-2-oxa-5-
azabicyclo[2.2.1]heptane (22). 5-Methylcytosine intermediate 20
(2.00 g, 3.50 mmol) was coevaporated with anhydrous pyridine (2 ×
10 mL) and then dissolved in anhydrous DCM (35 mL) and
anhydrous pyridine (1.4 mL, 17.3 mmol) under stirring at 0 °C.
Palmitoyl chloride (1.05 mL, 3.46 mmol) was added dropwise and the
resulting mixture stirred at 0 °C for 3 h. DCM (35 mL) was added and
the resulting mixture was washed consecutively with satd aq NaHCO3
(2 × 40 mL) and water (2 × 40 mL). The aqueous phases were back-
extracted with DCM (150 mL in total). The combined organic phase
was dried over Na2SO4, filtered and evaporated to dryness under
reduced pressure. The resulting residue was purified by silica gel
column chromatography (0−7% MeOH in DCM, v/v) to afford a
white foam (1.56 g, Rf = 0.5 (10% MeOH in DCM, v/v)), tentatively
assigned as (1R,3R,4R,7S)-1-(4,4′-Dimethoxytrityloxymethyl)-5-(hex-
adecanoyl)-7-hydroxy-3-(5-methyl-cytosine-1-yl)-2-oxa-5-azabicyclo-
[2.2.1]heptane (N2′-palmitoyl,5′-O-dimethoxytrityl-5-methylcytosine-
amino-LNA). A portion of this intermediate (78 mg, 5% of the total
amount) was dissolved in anhydrous DMF (2.0 mL). Anhydrous
pyridine (24 μL, 0.30 mmol) and benzoic anhydride (25 mg, 0.11
mmol) were added and the reaction mixture stirred at rt for 20 h.
Additional anhydrous pyridine (24 μL, 0.30 mmol) and benzoic
anhydride (29 mg, 0.13 mmol) were added, and the reaction mixture
stirred for another 78 h. The reaction mixture was diluted with EtOAc
(30 mL) and then successively washed with satd aq NaHCO3 (2 × 20
mL) and water (2 × 20 mL). The aqueous phases were back-extracted
with EtOAc (30 mL in total). The combined organic phase was dried
over Na2SO4, filtered and evaporated to dryness under reduced
pressure. The resulting residue was purified by silica gel column
chromatography (0−2% MeOH and 0.1% pyridine in DCM, v/v/v) to
afford a rotameric mixture of desired nucleoside 22 (71 mg, 45% from
20, 7:3 ratio according to 1H NMR) as a white foam. TLC (5%
MeOH in DCM, v/v): Rf = 0.4; 1H NMR (CDCl3) δ 13.43 (br s, 1H,
4-NH), 8.32−8.25 (m, 2HI, H2Bz, H6Bz), 8.17−8.23 (m, 2HII, H2Bz,
H6Bz), 7.84 (s, 1HI, H6), 7.77 (s, 1HII, H6), 7.55−7.22 (m, 12H,
13C NMR (DMSO-d6) δ 178.2 (COPh), 159.2 (C4), 158.3 (C4DMT
/
C4′DMT), 158.2 (C4DMT/C4′DMT), 155.0 (q, J = 36.5, COCF3,I), 154.8
(q, J = 37.0, COCF3,II), 146.9 (C2), 144.4 (C1″DMT), 137.1 (C1Bn,II),
137.0 (C1Bn,I), 136.7 (C6, C1Bz), 134.84 (C1DMT,I/C1′DMT,I), 134.83
(C1DMT,II/C1′DMT,II), 134.75 (C1DMT,II/C1′DMT,II), 134.73 (C1DMT,I
/
C1′DMT,I), 132.6 (C4Bz), 129.72 (C2DMT,II, C6DMT,II, C2′DMT,II
C6′DMT,II), 129.69 (C2DMT,I, C6DMT,I, C2′DMT,I, C6′DMT,I), 129.4
(C2Bz, C6Bz), 128.33 (C3Bz, C5Bz, C3Bn,I, C5Bn,I), 128.29 (C3Bn,II
,
,
C5Bn,II), 128.1 (C3″DMT, C5″DMT), 127.9 (C4Bn,I), 127.8 (C4Bn,II),
127.5 (C2″DMT, C6″DMT), 127.4 (C2Bn, C6Bn), 126.9 (C4″DMT), 115.8
(q, J = 288.5, CF3,II), 115.7 (q, J = 287.7, CF3,I), 113.40 (C3DMT,I
C5DMT,I/C3′DMT,I, C5′DMT,I), 113.38 (C3DMT,II, C5DMT,II/C3′DMT,II
,
,
C5′DMT,II), 113.36 (C3DMT,I, C5DMT,I/C3′DMT,I, C5′DMT,I), 113.35
(C3DMT,II, C5DMT,II/C3′DMT,II, C5′DMT,II), 109.4 (C5), 87.4 (C4′II),
86.3 (C1′II), 86.06 (CAr3,II), 86.03 (CAr3,I), 85.98 (C4′I), 75.8 (C3′I),
74.3 (C3′II), 71.4 (CH2PhII), 71.3 (CH2PhI), 61.3 (C2′I), 59.8 (C2′II),
58.5 (C5′I), 58.4 (C5′II), 55.0 (OCH3), 52.9 (C5″I), 52.8 (C5″II), 13.4
(5-CH3).52 19F NMR (DMSO-d6) δ −70.1 (CF3,II), −71.5 (CF3,I).
+
ESI-HRMS m/z 861.3104 ([M + H]+, C48H44F3N4O8 Calcd
861.3106).
(1R,3R,4R,7S)-7-Benzyloxy-1-(4,4′-dimethoxytrityl)oxymethyl-3-
(4-N-acetyl-5-methylcytosin-1-yl)-5-trifluoroacetyl-2-oxa-5-
azabicyclo[2.2.1]heptane (21b). Nucleoside 19 (1.00 g, 1.32 mmol)
was coevaporated with pyridine (3 mL) and then dissolved in a
mixture of anhydrous pyridine (7 mL) and anhydrous DCM (7 mL).
Ac2O (0.14 mL, 1.48 mmol) was added. The reaction mixture stirred
at rt for 24 h, whereafter Ac2O (0.07 mL, 0.74 mmol) and DMAP (79
mg, 0.65 mmol) were added, and the reaction mixture stirred for
additional 72 h. Ac2O (0.07 mL, 0.74 mmol) was added, and the
reaction mixture stirred for additional 24 h, whereafter the reaction
mixture was evaporated to dryness and the residue coevaporated with
toluene (3 × 10 mL). To the resulting residue was added DCM (50
mL) whereupon washing was performed with satd aq NaHCO3 (50
mL) and brine (50 mL). The combined aq phase was extracted with
DCM (25 mL), and the combined organic phase dried over MgSO4
and evaporated to dryness. The resulting residue was purified by
DCVC (0−6% MeOH in DCM, v/v) to afford a rotameric mixture of
the desired nucleoside 21b (868 mg, 82%, 2:3 ratio of rotamers
H2DMT, H6DMT, H2′DMT, H6′DMT, H2″DMT, H3″DMT, H4″DMT
H5″DMT, H6″DMT, H3Bz, H4Bz, H5Bz), 6.90−6.83 (m, 4H, H3DMT
,
,
H5DMT, H3′DMT, H5′DMT), 5.49 (s, 1H, H1′), 5.12 (s, 1HII, H2′), 4.52
(s, 1HI, H2′), 4.37 (s, 1HII, H3′), 4.29 (s, 1HI, H3′), 3.81−3.78 (m,
6H, 2 × OCH3), 3.73 (d, J = 11.0, 1HII, H5′A), 3.67 (d, J = 11.2, 1HI,
H5′A), 3.58−3.37 (m, 3H, H5′B, H5″A+B), 2.55−2.34 (m, 2HI,
COCH2), 2.12−2.19 (m, 2HII, COCH2), 1.82 (s, 3HI, 5-CH3), 1.76 (s,
3HII, 5-CH3), 1.72−1.49 (m, 2H, COCH2CH2), 1.38−1.12 (m, 24H,
(CH2)12CH3), 0.87 (t, J = 6.8, 3H, (CH2)14CH3). 13C NMR (CDCl3)
δ 179.7 (COPh), 173.3 (CO(CH2)14,I), 173.2 (CO(CH2)14,II), 159.9
(C4), 158.93 (C4DMT/C4′DMT), 158.87 (C4DMT/C4′DMT), 148.0 (C2),
1
according to H NMR) as a white foam. TLC (5% MeOH in EtOAc,
1
v/v): Rf (19) = 0.1, Rf (21b) = 0.6. H NMR (DMSO-d6) δ 9.90 (s,
1H, ex, 4-NH), 7.85−7.88 (m, 1H, H6), 7.36−7.40 (m, 2H, H2″DMT
H6″DMT), 7.23−7.43 (m, 10H, H3Bn, H4Bn, H5Bn, H2DMT, H6DMT
,
,
H2′DMT, H6′DMT, H3″DMT, H4″DMT, H5″DMT), 7.14−7.19 (m, 2H,
H2Bn, H6Bn), 6.85−6.91 (m, 4H, H3DMT, H5DMT, H3′DMT, H5′DMT),
5.75 (s, 1H, H1′), 5.30 (s, 1HII, H2′), 5.14 (s, 1HI, H2′), 4.61 (d, J =
11.8, 1HI, CH2PhA), 4.60 (d, J = 11.8, 1HII, CH2PhA), 4.54 (d, J = 11.8,
1H, CH2PhB), 4.45 (s, 1H, H3′), 3.74 (s, 3HI, OCH3), 3.733 (s, 3HII,
OCH3), 3.729 (s, 3HI, OCH3), 3.726 (s, 3HII, OCH3), 3.63−3.72 (m,
1H, H5″A), 3.45−3.55 (m, 3H, H5′A+B, H5″B), 2.30 (s, 3H, COCH3),
1.69 (s, 3H, 5-CH3). 13C NMR (DMSO-d6) δ 170.6 (COCH3,I), 170.5
144.5 (C1″DMT), 137.0 (C1Bz), 136.0 (C6), 135.6 (C1DMT,II
C1′DMT,II), 135.43 (C1DMT/C1′DMT), 135.40 (C1DMT,I/C1′DMT,I),
132.7 (C4Bz,I), 132.5 (C4Bz,II), 130.3 (C2DMT,I/II, C6DMT,I/II/C2′DMT,I/II
/
,
C6′DMT,I/II), 130.2 (C2DMT,I/II, C6DMT,I/II/C2′DMT,I/II, C6′DMT,I/II),
130.1 (C2Bz, C6Bz), 128.29 (C2″DMT,I/II, C3″DMT,I/II, C5″DMT,I/II
C6″DMT,I/II, C3Bz,I/II, C5Bz,I/II), 128.24 (C2″DMT,I/II, C3″DMT,I/II
C5″DMT,I/II, C6″DMT,I/II, C3Bz,I/II, C5Bz,I/II), 128.19 (C2″DMT,I/II
,
,
,
(COCH3,II), 162.7 (C4), 158.24 (C4DMT,I, C4′DMT,I), 158.20 (C4DMT,II
,
C4′DMT,II), 155.0 (q, J = 36.4, COCF3,I), 154.9 (q, J = 37.3, COCF3,II),
153.6 (C2I), 153.5 (C2II), 144.3 (C1″DMT), 140.7 (C6), 137.04
(C1Bn,II), 136.94 (C1Bn,I), 134.92 (C1DMT,I/C1′DMT,I), 134.83
C3″DMT,I/II, C5″DMT,I/II, C6″DMT,I/II, C3Bz,I/II, C5Bz,I/II), 127.4
(C4″DMT,I), 127.3 (C4″DMT,II), 113.58 (C3DMT,I, C5DMT,I/C3′DMT,I,
(C1DMT,II/C1′DMT,II), 134.82 (C1DMT,II/C1′DMT,II), 134.77 (C1DMT,I
/
C1′DMT,I), 129.7 (C2DMT, C6DMT, C2′DMT, C6′DMT), 128.31 (C3Bn,I
C5Bn,I), 128.28 (C3Bn,II, C5Bn,II), 128.0 (C3″DMT, C5″DMT), 127.9
(C4Bn,I), 127.8 (C4Bn,II), 127.6 (C2″DMT, C6″DMT), 127.51 (C2Bn,II
C6Bn,II), 127.50 (C2Bn,I, C6Bn,I), 126.9 (C4″DMT), 115.82 (q, J = 288.3,
CF3,II), 115.76 (q, J = 287.8, CF3,I), 113.40 (C3DMT,I, C5DMT,I
C3′DMT,I, C5′DMT,I), 113.38 (C3DMT,II, C5DMT,II/C3′DMT,II, C5′DMT,II),
,
C5′DMT,I), 113.56 (C3DMT, C5DMT/C3′DMT, C5′DMT), 113.52
(C3DMT,II, C5DMT.,II/C3′DMT,II, C5′DMT,II), 111.8 (C5), 89.1 (C4′II),
88.3 (C4′I), 87.7 (C1′I), 87.5 (C1′II), 87.1 (CAr3,I), 87.0 (CAr3,II),
70.3 (C3′I), 69.0 (C3′II), 63.5 (C2′I), 61.4 (C2′II), 59.6 (C5′II), 59.1
(C5′I), 55.4 (OCH3), 52.4 (C5″II), 51.1 (C5″I), 34.4 (COCH2,I), 34.1
(COCH2,II), 32.1 (CH2CH2CH3), 29.9, 29.8, 29.7, 29.63, 29.57, 29.5,
25.3 (COCH2CH2,I), 24.8 (COCH2CH2,II), 22.8 (CH2CH3), 14.3
((CH2)14CH3), 13.8 (5-CH3);53 HRMS-ESI m/z: 913.5085 ([M +
H]+, C55H68N4O8−H+ calcd 913.5110). When the benzoylation
reaction was run on 1.83 mmol scale, the overall yield from 20 to
,
/
113.36 (C3DMT,I, C5DMT,I/C3′DMT,I, C5′DMT,I), 113.35 (C3DMT,II
,
C5DMT,II/C3′DMT,II, C5′DMT,II), 105.5 (C5II), 105.4 (C5I), 87.3
(C4′II), 86.8 (C1′I), 86.4 (C1′II), 86.03 (CAr3,II), 85.99 (CAr3,I),
85.9 (C4′I), 75.6 (C3′I), 74.0 (C3′II), 71.4 (CH2PhII), 71.3 (CH2PhI),
10726
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Article
22 dropped to 32%, but no further optimization of the reaction
conditions was performed.
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Photochem. Rev. 2005, 6, 108−122.
(1R,3R,4R,7S)-7-(2-Cyanoethoxy(diisopropylamino)-
phosphinoxy)-1-(4,4′-dimethoxytrityloxymethyl)-5-(hexadecanoyl)-
7-hydroxy-3-(4-N-benzoyl-5-methyl-cytosine-1-yl)-2-oxa-5-
azabicyclo[2.2.1]heptane (23). Nucleoside 22 (530 mg, 0.58 mmol)
was coevaporated with anhydrous 1,2-DCE (2 × 5 mL) and
subsequently mixed with N,N-diisopropylammonium tetrazolide
(201 mg, 1.17 mmol). The solid mixture was dissolved in anhydrous
DCM (8 mL) under stirring and 2-cyanoethyl-N,N,N′,N′-tetraisopro-
pylphosphane (370 μL, 1.16 mmol) was added dropwise. The reaction
mixture was stirred at rt for 21 h whereupon EtOH (1 mL) was added
and the resulting mixture stirred for 40 min. The mixture was diluted
with DCM (25 mL), then washed with satd aq NaHCO3 (2 × 25 mL).
The combined aqueous phase was back-extracted with DCM (3 × 15
mL), and the combined organic phase dried over Na2SO4, filtered and
evaporated to dryness under reduced pressure. The resulting residue
was purified by silica gel column chromatography (0−100% EtOAc in
petroleum ether, v/v) to afford a rotameric mixture of diastereomeric
phosphoramidite 23 as a white foam (544 mg, 84%). Rf = 0.5 (50%
EtOAc in petroleum ether, v/v); 31P NMR (162 MHz, CDCl3) δP
149.90, 149.87, 149.4, 148.4; HRMS-ESI m/z: 1113.6143 ([M + H]+,
C64H85N6O9P−H+ calcd 1113.6188).
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ASSOCIATED CONTENT
* Supporting Information
■
S
(23) Socher, E.; Jarikote, D. V.; Knoll, A.; Roglin, L.; Burmeister, J.;
Seitz, O. Anal. Biochem. 2008, 375, 318−330.
Scheme S1. Synthesis of compounds 22 and 23. NMR data
(1H, 13C, 19F, and/or 31P) of compounds 12 and 16−23. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
■
*jwe@sdu.dk
Present Address
†Department of Chemistry, Technical University of Denmark,
2800 Kgs. Lyngby, Denmark.
(29) Johannsen, M. W.; Crispino, L.; Wamberg, M. C.; Kalra, N.;
Wengel, J. Org. Biomol. Chem. 2011, 9, 243−252.
(30) Babu, B. R.; Hrdlicka, P. J.; McKenzie, C. J.; Wengel, J. Chem.
Commun. 2005, 1705−1707.
Notes
The authors declare no competing financial interest.
(31) Kalek, M.; Madsen, A. S.; Wengel, J. J. Am. Chem. Soc. 2007,
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ACKNOWLEDGMENTS
■
(32) Kalek, M.; Benediktson, P.; Vester, B.; Wengel, J. Chem.
Commun. 2008, 762−764.
We greatly appreciate funding from Danish National Advanced
Technology Foundation and Danish National Research
Foundation. J.W. and A.S.J. acknowledges support from the
European Research Concil under the European Union’s
Seventh Framework program (FP7/2007-2013)/ERC Grant
agreement no. 268776.
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(52) Peaks for C2, C4, C5, 5-CH3, C6, COPh and C1Bz were broad
and of low intensity in 13C NMR, probably due to fast quadrupolar
relaxation via the nearby 14N-nuclei and rotameric change of the
benzamide bond. The peak for C1′I, which is of low intensity, probably
as a combination of quadrupolar relaxation and long distance coupling
with 19F, overlaps with CAr3 and C4′I peaks at 86.0 ppm.
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and of low intensity in 13C NMR, probably due to fast quadrupolar
relaxation via the nearby 14N-nuclei and rotameric change of the
benzamide bond.
10728
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